Sebastian Reichert

Survivin inhibition and DNA double-strand break repair: a molecular mechanism to overcome radioresistance in glioblastoma.

BACKGROUND AND PURPOSE Gliomas display prime examples of ionizing radiation (IR) resistant tumors. The IAP Survivin is reported to be critically involved in radiation resistance by anti-apoptotic and by caspase-independent mechanisms. The present study aimed to elucidate an interrelationship between Survivins cellular localization and DNA damage repair in glioma cells. MATERIAL AND METHODS Cellular distribution and nuclear complex formation were assayed by immunoblotting, immunofluorescence staining and co-immunoprecipitation of Survivin bound proteins in LN229 glioblastoma cells. Apoptosis induction, survival and DNA repair following IR were assayed by means of caspase3/7 activity, clonogenic assay, γ-H2AX/53BP1 foci formation, single cell gel electrophoresis assay, and DNA-PKcs kinase assay in the presence of Survivin siRNA or over expression of Survivin-GFP. RESULTS Following irradiation, we observed a nuclear accumulation and a direct interrelationship between Survivin, MDC1, γ-H2AX, 53BP1 and DNA-PKcs, which was confirmed by immunofluorescence co-localization. Survivin downregulation by siRNA resulted in an increased apoptotic fraction, decreased clonogenic survival and increased DNA-damage, as demonstrated by higher amount of DNA breaks and an increased amount of γ-H2AX/53BP1 foci post irradiation. Furthermore, we detected in Survivin-depleted LN229 cells a hampered S2056 (auto)phosphorylation and a significantly decreased DNA-PKcs kinase activity. CONCLUSION Nuclear accumulation of Survivin and interaction with components of the DNA-double-strand break (DSB) repair machinery indicates Survivin to regulate DSB damage repair that leads to a significant improvement of survival of LN229 glioblastoma cells.

Radiation-induced survivin nuclear accumulation is linked to DNA damage repair.

PURPOSE Increased expression of survivin has been identified as a negative prognostic marker in a variety of human cancers. We have previously shown that survivin is a radiation-resistance factor and that the therapeutic effect of survivin knock-down might result from an impaired DNA repair capacity. In this study, we aimed to elucidate an interrelationship between survivins cellular localization and DNA double-strand break repair. METHODS AND MATERIALS Survivins cellular distribution and nuclear complex formation were assayed by Western blotting of subcellular fractions, by immunofluorescence staining, and co-immunoprecipitation in SW480 colorectal cancer cells. DNA repair capacity was analyzed by kinetics of gamma-H2AX foci formation, and by DNA-dependent protein kinase (DNA-PKcs) assays in the presence of survivin-specific or nonspecific control siRNA. RESULTS Following irradiation, we observed a rapid nuclear accumulation of survivin and subsequent phosphorylation of the protein in the nucleus. Co-immunoprecipitation analyses from nuclear extracts revealed an interaction among survivin, Ku70, gamma-H2AX, MDC1, and DNA-PKcs that was confirmed by immunofluorescence co-localization in nuclear foci. Survivin knock down by siRNA resulted in an impaired DNA double strand break repair, as demonstrated by an increased detection of gamma-H2AX foci/nucleus at 60 min and a higher amount of residual gamma-H2AX foci at 24 hr postirradiation. Furthermore, we detected in survivin-depleted cells a hampered S2056 autophosphorylation of DNA-PKcs and a significantly decreased DNA-PKcs kinase activity. CONCLUSION These data indicate that nuclear survivin is linked to DNA double-strand break repair by interaction with members of the DNA double-strand breaks repair machinery, thus regulating DNA-PKcs activity.

A radiosensitizing effect of artesunate in glioblastoma cells is associated with a diminished expression of the inhibitor of apoptosis protein survivin

BACKGROUND AND PURPOSE Novel strategies to overcome an irradiation resistant phenotype may help to increase therapeutic efficacy in glioblastoma multiforme. The present study aimed to elucidate radiation sensitizing properties of artesunate, a semi synthetic derivate of artemisinin and to assess factors involved in this effect. MATERIALS AND METHODS LN229 and U87MG cells were treated with various concentrations of artesunate and radiation response was determined by a colony forming assay. Cell numbers, apoptosis induction, cell cycle distribution, and DNA repair following combined modality treatment were monitored by MTT-, caspase 3/7 assay, cytofluorometry, and γ-H2AX foci formation. Expression of survivin, survivin-GFP fusion protein, XIAP, cellular (c)IAP1 and cIAP2 was monitored by Western immunoblotting. RESULTS Treatment of glioma cells with artesunate and irradiation resulted in an increased apoptotic fraction, pronounced G2/M arrest and increased DNA damage as demonstrated by an elevated amount of γ-H2AX foci/nucleus. Incubation with artesunate lowers survivin expression in a time and dose-dependent manner, whereas expression of XIAP, cIAP1 and cIAP2 was not affected. In clonogenic assays, treatment with artesunate revealed a significantly reduced surviving fraction, whereas stable over expression of a survivin-GFP protein reversed artesunate-mediated radiosensitization. CONCLUSION Artesunate selectively down regulates survivin that contributes to a radio-sensitization of glioma cells by an increased induction of apoptosis, cell cycle arrest, and a hampered DNA damage response.

Targeting by cmHsp70.1-antibody coated and survivin miRNA plasmid loaded nanoparticles to radiosensitize glioblastoma cells.

Nanoparticles (NP) as carriers for anti-cancer drugs have shown great promise. Specific targeting of NP to malignant cells, however, remains an unsolved problem. Conjugation of antibodies specific for tumor membrane antigens to NP represents one approach to improve specificity and to increase therapeutic efficacy. In the present study, for the first time a novel membrane heat shock protein (Hsp70)-specific antibody (cmHsp70.1) was coupled to human serum albumin (HSA) NP, loaded with microRNA (miRNA) plasmids to target the inhibitor of apoptosis protein survivin. The physicochemical properties of monodisperse miRNA-loaded NP showed a diameter of 180 nm to 220 nm, a plasmid incorporation of more than 95% and a surface binding capacity of the antibody of 70-80%. Antibody-conjugated NP displayed an increased cellular uptake in U87MG and LN229 glioblastoma cells compared to isotype control antibody, PEG-coupled controls and peripheral blood lymphocytes (PBL). Survivin expression was significantly reduced in cells treated with the Hsp70-miRNA-NP as compared to non-conjugated NP. Hsp70-miRNA-NP enhanced radiation-induced increase in caspase 3/7 activity and decrease in clonogenic cell survival. In summary, cmHsp70.1 miRNA-NP comprise an enhanced tumor cell uptake and increased therapeutic efficacy of radiation therapy in vitro and provide the basis for the development of antibody-based advanced carrier systems for a tumor cell specific targeting.

Double targeting of Survivin and XIAP radiosensitizes 3D grown human colorectal tumor cells and decreases migration

BACKGROUND AND PURPOSE In the present study, we aimed to investigate the effect of single and double knockdown of the inhibitor of apoptosis proteins (IAP) Survivin and X-linked IAP (XIAP) on three-dimensional (3D) clonogenic survival, migration capacity and underlying signaling pathways. MATERIALS AND METHODS Colorectal cancer cell lines (HCT-15, SW48, SW480, SW620) were subjected to siRNA-mediated single or Survivin/XIAP double knockdown followed by 3D colony forming assays, cell cycle analysis, Caspase activity assays, migration assays, matrigel transmigration assays and Western blotting (Survivin, XIAP, Focal adhesion kinase (FAK), p-FAK Y397, Akt1, p-Akt1 S473, Extracellular signal-regulated kinase (ERK1/2), p-ERK1/2 T202/Y204, Glycogen synthase kinase (GSK)3β, p-GSK3β S9, nuclear factor (NF)-κB p65). RESULTS While basal cell survival was altered cell line-dependently, Survivin or XIAP single and Survivin/XIAP double knockdown enhanced cellular radiosensitivity of all tested cancer cell lines grown in 3D. Particularly double knockdown conditions revealed accumulation of cells in G2/M, increased subG1 fraction, elevated Caspase 3/7 activity, and reduced migration. Intracellular signaling showed dephosphorylation of FAK and Akt1 upon Survivin and/or Survivin/XIAP silencing. CONCLUSIONS Our results strengthen the notion of Survivin and XIAP to act as radiation resistance factors and further indicate that these apoptosis-regulating proteins are also functioning in cell cycling and cell migration.

Survivin-miRNA-loaded nanoparticles as auxiliary tools for radiation therapy: preparation, characterisation, drug release, cytotoxicity and therapeutic effect on colorectal cancer cells

One of the main challenges in radiation oncology is to overcome the resistance of cancer cells against treatment by molecular targeted approaches. Among the most promising targets is the inhibitor of apoptosis protein survivin, known to be associated with increased tumour aggressiveness and therapy resistance. The objective of this study was the development of a human serum albumin-based nanoparticulate carrier system for plasmid-mediated RNA interference (miRNA) and the investigation of its in vitro efficacy on survivin knockdown and cellular toxicity in SW480 colorectal cancer cells. The results demonstrate a robust nanoparticulate system of a size around 220 nm with a plasmid incorporation efficacy of about 90%. Moreover, treatment of carcinoma cells with survivin-miRNA nanoparticles resulted in reduction of survivin expression by 50% and increased cytotoxicity if combined with ionising irradiation. These nanoparticles comprise a promising option to enhance the response of carcinoma cells to therapy with ionising irradiation.

Hypoxia Enhances the Antiglioma Cytotoxicity of B10, a Glycosylated Derivative of Betulinic Acid

B10 is a glycosylated derivative of betulinic acid with promising activity against glioma cells. Lysosomal cell death pathways appear to be essential for its cytotoxicity. We investigated the influence of hypoxia, nutrient deprivation and current standard therapies on B10 cytotoxicity. The human glioma cell lines LN-308 and LNT-229 were exposed to B10 alone or together with irradiation, temozolomide, nutrient deprivation or hypoxia. Cell growth and viability were evaluated by crystal violet staining, clonogenicity assays, propidium iodide uptake and LDH release assays. Cell death was examined using an inhibitor of lysosomal acidification (bafilomycin A1), a cathepsin inhibitor (CA074-Me) and a short-hairpin RNA targeting cathepsin B. Hypoxia substantially enhanced B10-induced cell death. This effect was sensitive to bafilomycin A1 and thus dependent on hypoxia-induced lysosomal acidification. Cathepsin B appeared to mediate cell death because either the inhibitor CA074-Me or cathepsin B gene silencing rescued glioma cells from B10 toxicity under hypoxia. B10 is a novel antitumor agent with substantially enhanced cytotoxicity under hypoxia conferred by increased lysosomal cell death pathway activation. Given the importance of hypoxia for therapy resistance, malignant progression, and as a result of antiangiogenic therapies, B10 might be a promising strategy for hypoxic tumors like malignant glioma.