Katrin Manda

Immunomodulatory properties and molecular effects in inflammatory diseases of low-dose x-irradiation.

Inflammatory diseases are the result of complex and pathologically unbalanced multicellular interactions. For decades, low-dose X-irradiation therapy (LD-RT) has been clinically documented to exert an anti-inflammatory effect on benign diseases and chronic degenerative disorders. By contrast, experimental studies to confirm the effectiveness and to reveal underlying cellular and molecular mechanisms are still at their early stages. During the last decade, however, the modulation of a multitude of immunological processes by LD-RT has been explored in vitro and in vivo. These include leukocyte/endothelial cell adhesion, adhesion molecule and cytokine/chemokine expression, apoptosis induction, and mononuclear/polymorphonuclear cell metabolism and activity. Interestingly, these mechanisms display comparable dose dependences and dose-effect relationships with a maximum effect in the range between 0.3 and 0.7 Gy, already empirically identified to be most effective in the clinical routine. This review summarizes data and models exploring the mechanisms underlying the immunomodulatory properties of LD-RT that may serve as a prerequisite for further systematic analyses to optimize low-dose irradiation procedures in future clinical practice.

Effects of ionizing radiation on the immune system with special emphasis on the interaction of dendritic and T cells

Dendritic cells (DCs), as professional antigen-presenting cells, are members of the innate immune system and function as key players during the induction phase of adaptive immune responses. Uptake, processing, and presentation of antigens direct the outcome toward either tolerance or immunity. The cells of the immune system are among the most highly radiosensitive cells in the body. For high doses of ionizing radiation (HD-IR) both immune-suppressive effects after whole body irradiation and possible immune activation during tumor therapy were observed. On the other hand, the effects of low doses of ionizing radiation (LD-IR) on the immune system are controversial and seem to show high variability among different individuals and species. There are reports revealing that protracted LD-IR can result in radioresistance. But immune-suppressive effects of chronic LD-IR are also reported, including the killing or sensitizing of certain cell types. This article shall review the current knowledge of radiation-induced effects on the immune system, paying special attention to the interaction of DCs and T cells.

Cannabinoids increase lung cancer cell lysis by lymphokine-activated killer cells via upregulation of ICAM-1

Cannabinoids have been shown to promote the expression of the intercellular adhesion molecule 1 (ICAM-1) on lung cancer cells as part of their anti-invasive and antimetastatic action. Using lung cancer cell lines (A549, H460) and metastatic cells derived from a lung cancer patient, the present study addressed the impact of cannabinoid-induced ICAM-1 on cancer cell adhesion to lymphokine-activated killer (LAK) cells and LAK cell-mediated cytotoxicity. Cannabidiol (CBD), a non-psychoactive cannabinoid, enhanced the susceptibility of cancer cells to adhere to and subsequently be lysed by LAK cells, with both effects being reversed by a neutralizing ICAM-1 antibody. Increased cancer cell lysis by CBD was likewise abrogated when CBD-induced ICAM-1 expression was blocked by specific siRNA or by antagonists to cannabinoid receptors (CB1, CB2) and to transient receptor potential vanilloid 1. In addition, enhanced killing of CBD-treated cancer cells was reversed by preincubation of LAK cells with an antibody to lymphocyte function associated antigen-1 (LFA-1) suggesting intercellular ICAM-1/LFA-1 crosslink as crucial event within this process. ICAM-1-dependent pro-killing effects were further confirmed for the phytocannabinoid Δ(9)-tetrahydrocannabinol (THC) and R(+)-methanandamide (MA), a hydrolysis-stable endocannabinoid analogue. Finally, each cannabinoid elicited no significant increase of LAK cell-mediated lysis of non-tumor bronchial epithelial cells, BEAS-2B, associated with a far less pronounced (CBD, THC) or absent (MA) ICAM-1 induction as compared to cancer cells. Altogether, our data demonstrate cannabinoid-induced upregulation of ICAM-1 on lung cancer cells to be responsible for increased cancer cell lysis by LAK cells. These findings provide proof for a novel antitumorigenic mechanism of cannabinoids.

Cannabinoids inhibit angiogenic capacities of endothelial cells via release of tissue inhibitor of matrix metalloproteinases-1 from lung cancer cells.

Cannabinoids inhibit tumor neovascularization as part of their tumorregressive action. However, the underlying mechanism is still under debate. In the present study the impact of cannabinoids on potential tumor-to-endothelial cell communication conferring anti-angiogenesis was studied. Cellular behavior of human umbilical vein endothelial cells (HUVEC) associated with angiogenesis was evaluated by Boyden chamber, two-dimensional tube formation and fibrin bead assay, with the latter assessing three-dimensional sprout formation. Viability was quantified by the WST-1 test. Conditioned media (CM) from A549 lung cancer cells treated with cannabidiol, Δ(9)-tetrahydrocannabinol, R(+)-methanandamide or the CB2 agonist JWH-133 elicited decreased migration as well as tube and sprout formation of HUVEC as compared to CM of vehicle-treated cancer cells. Inhibition of sprout formation was further confirmed for cannabinoid-treated A549 cells co-cultured with HUVEC. Using antagonists to cannabinoid-activated receptors the antimigratory action was shown to be mediated via cannabinoid receptors or transient receptor potential vanilloid 1. SiRNA approaches revealed a cannabinoid-induced expression of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) as well as its upstream trigger, the intercellular adhesion molecule-1, to be causally linked to the observed decrease of HUVEC migration. Comparable anti-angiogenic effects were not detected following direct exposure of HUVEC to cannabinoids, but occurred after addition of recombinant TIMP-1 to HUVEC. Finally, antimigratory effects were confirmed for CM of two other cannabinoid-treated lung cancer cell lines (H460 and H358). Collectively, our data suggest a pivotal role of the anti-angiogenic factor TIMP-1 in intercellular tumor-endothelial cell communication resulting in anti-angiogenic features of endothelial cells.

Low dose effects of ionizing radiation on normal tissue stem cells.

In recent years, there has been growing evidence for the involvement of stem cells in cancer initiation. As a result of their long life span, stem cells may have an increased propensity to accumulate genetic damage relative to differentiated cells. Therefore, stem cells of normal tissues may be important targets for radiation-induced carcinogenesis. Knowledge of the effects of ionizing radiation (IR) on normal stem cells and on the processes involved in carcinogenesis is very limited. The influence of high doses of IR (>5Gy) on proliferation, cell cycle and induction of senescence has been demonstrated in stem cells. There have been limited studies of the effects of moderate (0.5-5Gy) and low doses (<0.5Gy) of IR on stem cells however, the effect of low dose IR (LD-IR) on normal stem cells as possible targets for radiation-induced carcinogenesis has not been studied in any depth. There may also be important parallels between stem cell responses and those of cancer stem cells, which may highlight potential key common mechanisms of their response and radiosensitivity. This review will provide an overview of the current knowledge of radiation-induced effects on normal stem cells, with particular focus on low and moderate doses of IR.

Omega-3 fatty acid supplementation in cancer therapy : does eicosapentanoic acid influence the radiosensitivity of tumor cells?

AbstractPurpose:The aim of this study was to evaluate whether the omega-3 polyunsaturated fatty acid cis-5,8,11,14,17-eicosapentanoic acid (EPA) can enhance the radiosensitivity of different human tumor cell lines.Materials and Methods:Colon adenocarcinoma cells HT-29, and two glioblastoma multiforme tumor cells T98G and U251 were cultured under standard conditions. Cell growth was observed during administration with different concentrations of EPA, using it as the free fatty acid dissolved in ethanol or bound to bovine serum albumin. To investigate the influence of EPA (free and bound) on radiosensitivity, tumor cells were pretreated 30 minutes or 24 hours prior to irradiation with the fatty acid. Cell survival was measured by colony-forming assays.Results:When combined with irradiation, incubation with EPA was found to result in enhanced radiosensitivity with substantial variation: while there was strong radiosensitization for HT-29 and U251 cells, almost no effect for T98G cells was observed. A marked radiosensitization was clearly dependent on the treatment schedule.Conclusion:The observations suggest that EPA is not only a nutritional adjuvant but also may be a potential candidate to enhance the efficacy of irradiation on human cancer cells.ZusammenfassungZiel:In der vorliegenden Arbeit wurde geprüft, ob die polyungesättigte Omega-3-Fettsäure cis-5,8,11,14,17-Eicosapentaensäure (EPA) die Strahlensensibilität verschiedener humaner Tumorzelllinien erhöhen kann.Methodik:Zellen eines kolorektalen Adenokarzinoms (HT-29) sowie zweier Glioblastome (T98G und U251) wurden unter Standardbedingungen kultiviert. Um den Einfluss von EPA auf das Wachstum der Zellen zu ermitteln, wurden diese verschiedenen EPAKonzentrationen ausgesetzt. Dabei erfolgte die Zugabe von EPA entweder als freie Fettsäure (gelöst in Ethanol) oder gebunden an Albumin. Zur Untersuchung der Wirkung von EPA (frei und gebunden) auf die Strahlensensibilität der Tumorzellen wurden die Zellen 30 min bzw. 24 h vor der Bestrahlung mit der Fettsäure behandelt und das Zellüberleben anhand von Koloniebildungstests ermittelt.Ergebnisse:Die zusätzliche Behandlung der Zellen mit EPA vor der Bestrahlung resultierte in einer unterschiedlich stark ausgeprägten Erhöhung der Radiosensitivität der Tumorzellen: Während für die HT-29- and U251-Zellen eine deutliche Strahlensensibilisierung nachweisbar war, konnte bei den T98G-Zellen kein Effekt verzeichnet werden. Die Strahlensensibilisierung war abhängig vom Zeitpunkt der EPA-Zugabe.Zusammenfassung:Die erhaltenen Ergebnisse zeigen, dass die Omega-3-Fettsäure EPA in der Tumortherapie nicht nur als Nahrungsergänzungsmittel von Bedeutung ist, sondern möglicherweise auch zur Wirkungssteigerung der Bestrahlung auf Tumorzellen beitragen kann.

Omega-3 Fatty Acid Supplementation in Cancer Therapy

AbstractPurpose:The aim of this study was to evaluate whether the omega-3 polyunsaturated fatty acid cis-5,8,11,14,17-eicosapentanoic acid (EPA) can enhance the radiosensitivity of different human tumor cell lines.Materials and Methods:Colon adenocarcinoma cells HT-29, and two glioblastoma multiforme tumor cells T98G and U251 were cultured under standard conditions. Cell growth was observed during administration with different concentrations of EPA, using it as the free fatty acid dissolved in ethanol or bound to bovine serum albumin. To investigate the influence of EPA (free and bound) on radiosensitivity, tumor cells were pretreated 30 minutes or 24 hours prior to irradiation with the fatty acid. Cell survival was measured by colony-forming assays.Results:When combined with irradiation, incubation with EPA was found to result in enhanced radiosensitivity with substantial variation: while there was strong radiosensitization for HT-29 and U251 cells, almost no effect for T98G cells was observed. A marked radiosensitization was clearly dependent on the treatment schedule.Conclusion:The observations suggest that EPA is not only a nutritional adjuvant but also may be a potential candidate to enhance the efficacy of irradiation on human cancer cells.ZusammenfassungZiel:In der vorliegenden Arbeit wurde geprüft, ob die polyungesättigte Omega-3-Fettsäure cis-5,8,11,14,17-Eicosapentaensäure (EPA) die Strahlensensibilität verschiedener humaner Tumorzelllinien erhöhen kann.Methodik:Zellen eines kolorektalen Adenokarzinoms (HT-29) sowie zweier Glioblastome (T98G und U251) wurden unter Standardbedingungen kultiviert. Um den Einfluss von EPA auf das Wachstum der Zellen zu ermitteln, wurden diese verschiedenen EPAKonzentrationen ausgesetzt. Dabei erfolgte die Zugabe von EPA entweder als freie Fettsäure (gelöst in Ethanol) oder gebunden an Albumin. Zur Untersuchung der Wirkung von EPA (frei und gebunden) auf die Strahlensensibilität der Tumorzellen wurden die Zellen 30 min bzw. 24 h vor der Bestrahlung mit der Fettsäure behandelt und das Zellüberleben anhand von Koloniebildungstests ermittelt.Ergebnisse:Die zusätzliche Behandlung der Zellen mit EPA vor der Bestrahlung resultierte in einer unterschiedlich stark ausgeprägten Erhöhung der Radiosensitivität der Tumorzellen: Während für die HT-29- and U251-Zellen eine deutliche Strahlensensibilisierung nachweisbar war, konnte bei den T98G-Zellen kein Effekt verzeichnet werden. Die Strahlensensibilisierung war abhängig vom Zeitpunkt der EPA-Zugabe.Zusammenfassung:Die erhaltenen Ergebnisse zeigen, dass die Omega-3-Fettsäure EPA in der Tumortherapie nicht nur als Nahrungsergänzungsmittel von Bedeutung ist, sondern möglicherweise auch zur Wirkungssteigerung der Bestrahlung auf Tumorzellen beitragen kann.

Investigation of Epothilone B-Induced Cell Death Mechanisms in Human Epithelial Cancer Cells –in Consideration of Combined Treatment With Ionizing Radiation

Epothilone B was shown to have promising chemo- and radiosensitizing effects on cells, but the mechanisms underlying cell death remain ambiguous. The aim of the study was to examine selected cell death pathways on the basis of FaDu and A549 cells. Western blot analyses were used for investigation of specific apoptotic markers. Immunofluorescence imaging and flow cytometry were utilized for examination of cell death mechanisms. DNA-staining was used for studying influence of epothilone B on micronucleus rate. We showed that epothilone B can initiate cell death via apoptosis and mitotic catastrophe, but induction of cell death was cell type specific.

Immunomodulatory Properties of low-dose ionizing Radiation on Human Endothelial Cells

Abstract Purpose: The application of radiation therapy (RT) is not only used to treat cancer, in Germany, it is also an accepted and empirically established treatment of patients with benign diseases at low doses. The immune modulatory response generated by low-dose RT has a supporting anti-inflammatory effect within the treatment of inflammation-related diseases. The aim of this study was to investigate the effect of ionizing radiation (IR) on the expression and secretion of inflammatory mediators by endothelial cells (ECs) exposed to low and moderate doses. Methods: Non-activated and activated EC were irradiated with doses between 0.01 Gy and 2 Gy with X-rays. Using a multiplex-assay, protein values of interleukin-8 (IL-8), granulocyte macrophage colony-stimulating factor (G-CSF) and platelet-derived growth factor (PDGF-BB) were measured in the supernatant at different time-points. To investigate possible differences between mRNA expression and protein secretion after IR, the mRNA expression of IL-8, G-CSF and PDGF-BB was determined by real-time quantitative PCR. Results: Radiation treatment caused non-linear dose dependent effects on pro-inflammatory cytokine secretion of IL-8; G-CSF and PDGF-BB. The mRNA-expression levels of those cytokines were non-linear dose-dependent and differed from protein level in the culture supernatant. Conclusions: This study provides deeper insights into the radiobiological effects of radiation doses below 0.3 Gy, in particular 0.05 Gy, and their significant immunomodulatory properties on EC, which is very important in order to assess the effect of LD-IR on EC.

Effect of epothilone B on cell cycle, metabolic activity, and apoptosis induction on human epithelial cancer cells-under special attention of combined treatment with ionizing radiation.

In recent studies, epothilone B was shown to have a cytotoxic and radiosensitizing effect on cells. The aim of our investigation was to explain this impact by examining the mode of action of epothilone B on FaDu and A549 tumor cells. Flow cytometry was used for cell cycle distribution and for the evaluation of apoptosis. Metabolic activity was studied by proliferation assay. Influence on nuclei morphology was investigated by DNA-staining. We showed that epothilone B-induced G2/M accumulation is the main rationale for drug-induced radiosensitivity. The cytotoxic effect resulted in apoptotic cell death, decreased metabolic activity, and formation of multinucleated cells.