Targeting neovascularization and respiration of tumor grafts grown on chick embryo chorioallantoic membranes.

Since growing tumors stimulate angiogenesis, via vascular endothelial growth factor (VEGF), angiogenesis inhibitors (AIs, blockers of the VEGF signaling pathway) have been introduced to cancer therapy. However, AIs often yielded only modest and short-lived gains in cancer patients and more invasive tumor phenotypes in animal models. Combining anti-VEGF strategies with lactate uptake blockers may boost both efficacy and safety of AIs. We assessed this hypothesis by using the ex ovo chorioallantoic membrane (CAM) assay. We show that AI-based monotherapy (Avastin®, AVA) increases tumor hypoxia in human CAM cancer cell xenografts and cell spread in human as well as canine CAM cancer cell xenografts. In contrast, combining AVA treatment with lactate importer MCT1 inhibitors (α-cyano-4-hydroxycinnamic acid (CHC) or AZD3965 (AZD)) reduced both tumor growth and cell dissemination of human and canine explants. Moreover, combining AVA+AZD diminished blood perfusion and tumor hypoxia in human explants. Thus, the ex ovo CAM assay as an easy, fast and cheap experimental setup is useful for pre-clinical cancer research. Moreover, as an animal-free experimental setup the CAM assay can reduce the high number of laboratory animals used in pre-clinical cancer research.

Dynamic In Vivo Profiling of DNA Damage and Repair after Radiotherapy Using Canine Patients as a Model.

Time resolved data of DNA damage and repair after radiotherapy elucidates the relation between damage, repair, and cell survival. While well characterized in vitro, little is known about the time-course of DNA damage response in tumors sampled from individual patients. Kinetics of DNA damage after radiotherapy was assessed in eight dogs using repeated in vivo samples of tumor and co-irradiated normal tissue analyzed with comet assay and phosphorylated H2AX (γH2AX) immunohistochemistry. In vivo results were then compared (in silico) with a dynamic mathematical model for DNA damage formation and repair. Maximum %DNA in tail was observed at 15-60 min after irradiation, with a rapid decrease. Time-courses of γH2AX-foci paralleled these findings with a small time delay and were not influenced by covariates. The evolutionary parameter search based on %DNA in tail revealed a good fit of the DNA repair model to in vivo data for pooled sarcoma time-courses, but fits for individual sarcoma time-courses suffer from the heterogeneous nature of the in vivo data. It was possible to follow dynamics of comet tail intensity and γH2AX-foci during a course of radiation using a minimally invasive approach. DNA repair can be quantitatively investigated as time-courses of individual patients by integrating this resulting data into a dynamic mathematical model.

AMPK-independent autophagy promotes radioresistance of human tumor cells under clinical relevant hypoxia in vitro.

BACKGROUND AND PURPOSE: Blocking of the autophagy-signaling has the potential to improve cancer therapy. In the present study, the role of autophagy for radioresistance of human tumor cells was tested under clinically relevant hypoxia (1% O2). MATERIALS AND METHODS: Non-small cell lung cancer cell lines A549 and H460, head and neck squamous cell carcinoma FaDu, colon carcinoma cell line HCT116 and mouse-embryo-fibroblasts were analyzed under normoxic (21% O2) and hypoxic (0.01% and 1% O2) conditions with respect to clonogenic cell survival and hypoxia-induced autophagy. Immunofluorescence and electron microscopy were used to monitor the autophagy process and Western blotting of LC3, AMPK, and BNIP3 was applied to analyze autophagy signaling. RESULTS: Clinically relevant hypoxia stimulated autophagy in tumor cells as indicated by enhanced LC3-I to LC3-II conversion. Furthermore, hypoxia stimulated autophagy was approved by Immunofluorescence staining and electron-microscopy analysis of autophagosome vacuoles. Preconditioning of tumor cells to moderate-hypoxia increased their radioresistance that was significantly reversed following pretreatment with autophagy inhibitor, chloroquine. Using siRNA against AMPK as well as AMPK deficient cells, autophagy stimulation by 1% O2 was shown to be AMPK-independent. However, a correlation between the expression of BNIP3 and autophagy-stimulation was observed under this condition. CONCLUSION: Under clinically relevant hypoxia (1% O2) the stimulation of autophagy mediates resistance of hypoxic tumor cells to ionizing radiation, which is independent of AMPK signaling.

Cisplatin-mediated radiosensitization of non-small cell lung cancer cells is stimulated by ATM inhibition.

BACKGROUND AND PURPOSE: Cisplatin activates ataxia-telangiectasia-mutated (ATM), a protein with roles in DNA repair, cell cycle progression and autophagy. We investigated the radiosensitizing effect of cisplatin with respect to its effect on ATM pathway activation. MATERIAL AND METHODS: Non-small cell lung cancer cells (NSCLC) cell lines (A549, H460) and human fibroblast (ATM-deficient AT5, ATM-proficient 1BR3) cells were used. The effects of cisplatin combined with irradiation on ATM pathway activity, clonogenicity, DNA double-strand break (DNA-DSB) repair and cell cycle progression were analyzed with Western blotting, colony formation and γ-H2AX foci assays as well as FACS analysis, respectively. RESULTS: Cisplatin radiosensitized H460 cells, but not A549 cells. Radiosensitization of H460 cells was not due to impaired DNA-DSB repair, increased apoptosis or cell cycle dysregulation. The lack of radiosensitization demonstrated for A549 cells was associated with cisplatin-mediated stimulation of ATM (S1981) and AMPKα (T172) phosphorylation and autophagy. However, in both cell lines inhibition of ATM and autophagy by KU-55933 and chloroquine diphosphate (CQ) respectively resulted in a significant radiosensitization. Combined treatment with the AMPK inhibitor compound-C led to radiosensitization of A549 but not of H460 cells. As compared to the treatment with KU-55933 alone, radiosensitivity of A549 cells was markedly stimulated by the combination of KU-55933 and cisplatin. However, the combination of CQ and cisplatin did not modulate the pattern of radiation sensitivity of A549 or H460 cells. In accordance with the results that cisplatin via stimulation of ATM activity can abrogate its radiosensitizing effect, ATM deficient cells were significantly sensitized to ionizing radiation by cisplatin. CONCLUSION: The results obtained indicate that ATM targeting can potentiate cisplatin-induced radiosensitization.

Autophagy contributes to resistance of tumor cells to ionizing radiation.

BACKGROUND AND PURPOSE: Autophagy signaling is a novel important target to improve anticancer therapy. To study the role of autophagy on resistance of tumor cells to ionizing radiation (IR), breast cancer cell lines differing in their intrinsic radiosensitivity were used. MATERIALS AND METHODS: Breast cancer cell lines MDA-MB-231 and HBL-100 were examined with respect to clonogenic cell survival and induction of autophagy after radiation exposure and pharmacological interference of the autophagic process. As marker for autophagy the appearance of LC3-I and LC3-II proteins was analyzed by SDS-PAGE and Western blotting. Formation of autophagic vacuoles was monitored by immunofluorescence staining of LC3. RESULTS: LC3-I and LC3-II formation differs markedly in radioresistant MDA-MB-231 versus radiosensitive HBL-100 cells. Western blot analyses of LC3-II/LC3-I ratio indicated marked induction of autophagy by IR in radioresistant MDA-MB-231 cells, but not in radiosensitive HBL-100 cells. Indirect immunofluorescence analysis of LC3-II positive vacuoles confirmed this differential effect. Pre-treatment with 3-methyladenine (3-MA) antagonized IR-induced autophagy. Likewise, pretreatment of radioresistant MDA-231 cells with autophagy inhibitors 3-MA or chloroquine (CQ) significantly reduced clonogenic survival of irradiated cells. CONCLUSION: Our data clearly indicate that radioresistant breast tumor cells show a strong post-irradiation induction of autophagy, which thus serves as a protective and pro-survival mechanism in radioresistance.

Autophagy is activated by proteasomal inhibition and involved in aggresome clearance in cultured astrocytes.

A common pathway underlying a variety of neurodegenerative disorders is the aggregation and deposition of misfolded proteins. Proteasomal inhibition has been demonstrated to promote the formation of intracellular inclusions. We have shown before that astrocytes respond to the treatment with the proteasome inhibitor MG-132 by aggresome formation and cytoskeletal disturbances, but unlike oligodendrocytes do not die by apoptotic cell death and have the capability to recover. This study was undertaken to elucidate if the autophagy-lysosomal pathway participates in the efficient recovery process in astrocytes and is modulated under conditions of proteasomal inhibition. The data show that the autophagic pathway was stimulated during a 24-h treatment with the proteasome inhibitor MG-132 in a time and concentration-dependent manner. It remained at an elevated level throughout a 24-h recovery period in the absence of MG-132 and participates in the aggregate clearing process. In the presence of the specific inhibitor of macroautophagy, 3-methyladenine, cell viability was impaired, aggregates were not as efficiently removed and HSP25, αB-crystallin and ubiquitinated proteins remained in the insoluble protein fraction. LC3-II positive puncta, indicative of autophagosomes, were formed abundantly in the cells after proteasome inhibition and were seen in close association with the aggregates. Hence, the ability of astrocytes to upregulate autophagic degradation might contribute to their resistance against proteasomal stress situations and act as a compensatory mechanism when the proteasome is impaired.