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// Peter Hambsch 1 , Yury P. Istomin 3 , Dimitri A. Tzerkovsky 3 , Ina Patties 1 , Jochen Neuhaus 4 , Rolf-Dieter Kortmann 1 , Stanislav Schastak 2 and Annegret Glasow 1 1 Department of Radiation Therapy, University of Leipzig, 04103 Leipzig, Germany 2 Department of Ophthalmology, University of Leipzig, 04103 Leipzig, Germany 3 N. N. Alexandrov Republican Scientific and Practical Center of Oncology and Medical Radiology, 223040 Lesnoy, Republic of Belarus 4 Department of Urology, University of Leipzig, 04103 Leipzig, Germany Correspondence to: Annegret Glasow, email: Annegret.Glasow@medizin.uni-leipzig.de Keywords: photodynamic therapy, glioblastoma, THPTS, combination, ionizing radiation Received: January 10, 2017 Accepted: August 04, 2017 Published: August 23, 2017 ABSTRACT Background: So far, glioblastomas cannot be cured by standard therapy and have an extremely poor median survival of about 15 months. The photodynamic therapy (PDT) with next generation photosensitizers, reaching a higher therapeutic depth, might offer a new, adjuvant treatment strategy in brain cancer therapy. Here, we investigated the effect of THPTS-PDT combined with ionizing irradiation (IR) on glioblastoma cells in vitro and in vivo . Results: THPTS colocalized to mitochondria and was not found in the nucleus. THPTS (2–20 μg/ml)-PDT significantly reduced the proliferation, metabolic activity and clonogenic survival and induced cell death mainly through apoptosis and autophagy. THPTS-PDT combined with IR decreased the clonogenicity significantly compared to single treatments. THPTS (≤ 300 μg/ml) alone showed no dark toxicity. The maximum therapeutic depth of THPTS-PDT in C6 glioblastomas was 13 mm. Materials and Methods: Three human glioblastoma cell lines (U-87 MG, A-172, DBTRG-05MG) were incubated with THPTS (1–300 μg/ml) 3–24 hours before laser treatment (760 nm, 30 J/cm²). THPTS localization and effects on metabolic activity, proliferation, cell death mechanisms and long-term reproductive survival were assessed. IR was conducted on an X-ray unit (0.813 Gy/min). Results were verified in vivo on a subcutaneous C6 glioblastoma model in Wistar rats. Conclusions: This study demonstrated efficient THPTS-PDT in glioblastoma cells, in vitro and in vivo . The combinatorial effects of THPTS-PDT and IR are of specific clinical interest as enhanced eradication of infiltrating glioblastoma cells in the tumor surrounding tissue might possibly reduce the commonly occurring local relapses.

Background Glioblastoma is the most common and aggressive brain tumour in adults with a median overall survival of only 14 months after standard therapy with radiation therapy (IR) and temozolomide (TMZ). In a novel multimodal treatment approach we combined the checkpoint kinase 1 (Chk1) inhibitor SAR-020106 (SAR), disrupting homologue recombination, with standard DNA damage inducers (IR, TMZ) and the epigenetic/cytotoxic drug decitabine (5-aza-2′-deoxycitidine, 5-aza-dC). Different in vitro glioblastoma models are monitored to evaluate if the impaired DNA damage repair may chemo/radiosensitize the tumour cells. Methods Human p53-mutated (p53-mut) and -wildtype (p53-wt) glioblastoma cell lines (p53-mut: LN405, T98G; p53-wt: A172, DBTRG) and primary glioblastoma cells (p53-mut: P0297; p53-wt: P0306) were treated with SAR combined with TMZ, 5-aza-dC, and/or IR and analysed for induction of apoptosis (AnnexinV and sub-G1 assay), cell cycle distribution (nuclear PI staining), DNA damage (alkaline comet or gH2A.X assay), proliferation inhibition (BrdU assay), reproductive survival (clonogenic assay), and potential tumour stem cells (nestinpos/GFAPneg fluorescence staining). Potential treatment-induced neurotoxicity was evaluated on nestin-positive neural progenitor cells in a murine entorhinal-hippocampal slice culture model. Results SAR showed radiosensitizing effects on the induction of apoptosis and on the reduction of long-term survival in p53-mut and p53-wt glioblastoma cell lines and primary cells. In p53-mut cells, this effect was accompanied by an abrogation of the IR-induced G2/M arrest and an enhancement of IR-induced DNA damage by SAR treatment. Also TMZ and 5-aza-dC acted radioadditively albeit to a lesser extent. The multimodal treatment achieved the most effective reduction of clonogenicity in all tested cell lines and did not affect the ratio of nestinpos/GFAPneg cells. No neurotoxic effects were detected when the number of nestin-positive neural progenitor cells remained unchanged after multimodal treatment. Conclusion The Chk1 inhibitor SAR-020106 is a potent sensitizer for DNA damage-induced cell death in glioblastoma therapy strongly reducing clonogenicity of tumour cells. Selectively enhanced p53-mut cell death may provide stronger responses in tumours defective of non-homologous end joining (NHEJ). Our results suggest that a multimodal therapy involving DNA damage inducers and DNA repair inhibitors might be an effective anti-tumour strategy with a low risk of neurotoxicity.

Radiation-induced heart disease represents a late complication of thoracic radiotherapy. We investigated the inflammatory and thrombotic response after local heart irradiation in wild-type and atherosclerosis-prone mice.Atherosclerosis-prone ApoE(-/-) and C57BL/6 wild-type mice were sacrificed 20, 40, and 60 weeks after irradiation with 0.2, 2, 8, or 16 Gy. The expression of CD31, vascular cell adhesion molecule-1 (VCAM-1), thrombomodulin (TM), and CD45 were quantified by immunofluorescence staining of heart tissue sections.Microvascular density decreased at 40 weeks after 16 Gy in C57BL/6 but not in ApoE(-/-) mice. CD31 expression declined in C57BL/6 mice at 40 weeks (8 Gy), but increased in ApoE(-/-) mice at 20 (2/8/16 Gy) and 60 weeks (16 Gy). Capillary area decreased in C57BL/6 at 40 weeks (8/16 Gy) but increased in ApoE(-/-) mice at 20 weeks (16 Gy). Endocardial VCAM-1 expression remained unchanged. TM-positive capillaries decreased at 40 weeks (8/16 Gy) in C57BL/6 and at 60 weeks (2/16 Gy) in ApoE(-/-) mice. Leukocyte infiltration transiently rose 40 weeks after 8 Gy (only ApoE(-/-)) and 16 Gy. After receiving a low irradiation dose of 0.2 Gy, no significant changes were observed in any of the mouse models.This study demonstrated that local heart irradiation affects microvascular structure and induces inflammatory/thrombotic responses in mice in a dose- and time-dependent manner. Thereby, significant prothrombotic changes were found in both strains, although they were progressive in ApoE(-/-) mice only. Proinflammatory responses, like the increase of adhesion molecules and leukocyte infiltration, were more pronounced and occurred at lower doses in ApoE(-/-) vs. C57BL/6 mice. These findings indicate that metabolic risk factors, such as decreased ApoE lipoproteins, may lead to an enhanced proinflammatory and prothrombotic late response in locally irradiated hearts.

BACKGROUND AND OBJECTIVE: In recent years, the natural plant polyphenol resveratrol (RV) attracted an increasing attention in tumor therapy. Currently, clinical trials for colon cancer and multiple myeloma have been completed. RV has to been shown to exhibit tumor preventive as well as anticancer effects. These are of particular interest for the therapy of pediatric medulloblastoma (MB), as low-risk MB patients (5-year survival up to 95 %) require a therapy focused on the reduction of late adverse effects (i.e. neurological and endocrine disorders), whereas patients belonging to the high-risk group (i.e. such owning MYC amplification; 5-year survival 13 %) urgently need a more intensive therapy to improve survival. In the present study, we analyzed RV-mediated changes on the clonogenic survival and proliferation of irradiated medulloblastoma cells. Furthermore, we investigated if RV may induce double-strand breaks (DSB), and if combined RV/irradiation (IR) treatment affects the number of potential tumor stem cells (TSC). METHODS: Three MB cell lines were treated with RV doses ranged from 9.5 µM to 40 µM (based on preliminary experiments examining metabolic activity or clonogenic survival of each cell line) for three or six days. 2 Gy IR was applied immediately before RV (3 d treatment) or after three days (6 d treatment). Clonogenic assays were conducted at the end of treatment time. DSB were measured by γH2AX assay 1 h and 24 h after IR or RV. To quantify potential TSC, we detected vital CD15+ and CD133+ cells by flow cytometry three days after IR and RV treatment. Effects on proliferation were measured flow cytometrically in CSFE-labeled cells. RESULTS: The post-treatment of irradiated MB cells with RV for three days significantly decreased the clonogenic survival 1.8-fold (DAOY) to 36-fold (MEB-Med8a). The longer RV treatment period reached stronger effects decreasing the clonogenic survival by up to 900-fold in irradiated D283-Med cells. Both, IR and RV alone, induced DSB. An increase of residual, unrepaired IR-induced DSB was observed after RV treatment in two-out-of-three MB cell lines (DAOY, MEB-Med8a). Examining the number of potential TSC, we revealed an enrichment of CD15+ and CD133+ cells after RV or IR alone, which was slightly enhanced by combination of both. We found no differences in proliferation rates between TSC and non-TSC. Therefore, we assume that the enrichment of TSC is caused by a higher treatment resistance of potential TSC compared to non-TSC. CONCLUSION: RV may enhance the radiation effects on clonogenic survival of human MB cells, possibly through an increased induction of additional DSB. Further studies in animal MB models are warranted to clarify if the observed in vitro effects translate into improved survival rates in vivo.

Background Medulloblastoma (MB) is the most common malignant brain tumor in childhood with a 5-year survival of approximately 60%. We have recently shown that treatment of human MB cells with 5-aza-2’-deoxycytidine (5-aza-dC) reduces the clonogenic survival significantly. Here, we tested combinatorial effects of 5-aza-dC with other epigenetic (valproic acid, SAHA) and differentiation-inducing drugs (resveratrol, abacavir, retinoic acid) on human MB cells in vitro to intensify the antitumor therapy further. Methods Three human MB cell lines were treated with 5-aza-dC alone or in combination for three or six days. Metabolic activity was measured by WST-1 assay. To determine long-term reproductive survival, clonogenic assays were performed. Induction of DNA double-strand break (DSB) repair was measured by γH2AX assay. Results The applied single drugs, except for ATRA, reduced the metabolic activity dose-dependently in all MB cell lines. Longer treatment times enhanced the reduction of metabolic activity by 5-aza-dC. Combinatorial treatments showed differential, cell line-dependent responses indicating an important impact of the genetic background. 5-Aza-dC together with resveratrol was found to exert the most significant inhibitory effects on metabolic activity in all cell lines. 5-aza-dC alone reduced the clonogenicity of MB cells significantly and induced DSB with no further changes after adjuvant administration of resveratrol. Conclusion The observed significant decrease in metabolic activity by combinatorial treatment of MB cells with 5-aza-dC and resveratrol does not translate into long-term reproductive survival deficiency in vitro. Further studies in animal models are needed to clarify the resveratrol-mediated anticancer mechanisms in vivo.

In recent years, epigenetic modulators were introduced into tumor therapy. Here, the authors investigated the antitumor effect of 5-aza-2'-deoxycytidine-(5-aza-dC-)induced demethylation combined with irradiation on human medulloblastoma (MB) cells, which form the most common malignant brain tumor in children.Three MB cell lines were treated with 5-aza-dC in a low-dose (0.1 microM, 6 days) or high-dose (3/5 microM, 3 days) setting and irradiated with 2, 4, 6, or 8 Gy single dose on an X-ray unit. Methylation status and mRNA expression of three candidate genes were analyzed by methylation-specific PCR (polymerase chain reaction) and quantitative real-time RT-PCR. Cell survival and mortality were determined by trypan blue exclusion test. Proliferation was analyzed by BrdU incorporation assay, and long-term cell survival was assessed by clonogenic assay.5-aza-dC treatment resulted in partial promoter demethylation and increased expression of hypermethylated candidate genes. A significant decrease of vital cell count, proliferation inhibition and increase of mortality was observed in 5-aza-dC-treated as well as in irradiated MB cells, whereby combination of both treatments led to additive effects. Although high-dose 5-aza-dC treatment was more effective in terms of demethylation, clonogenic assay revealed no differences between high- and low-dose settings indicating no relevance of 5-aza-dC-induced demethylation for decreased cell survival. MB cells pretreated with 5-aza-dC showed significantly lower plating efficiencies than untreated cells at all irradiation doses investigated. Analysis of surviving curves in irradiated MB cells, however, revealed no significant differences of alpha-, beta-values and 2-Gy surviving fraction with or without 5-aza-dC treatment.5-aza-dC did not enhance radiation sensitivity of MB cells but significantly reduced the clonogenicity versus irradiation alone, which merits further investigation of its potential clinical application in MB possibly by combination with other chemotherapeutic agents.

BACKGROUND AND OBJECTIVE: Radiation therapy plays an essential role in the treatment of primary or metastatic CNS malignancies, but radiation-induced adverse effects remain a significant risk. Radiation-induced damage of the neural stem cell pool in the hippocampus is thought to be one of the major reasons for the long-term decline in neurocognitive performance, especially in pediatric patients. Here, we investigate the short- and long-term dose-response relationship of neurogenesis, covering a dose range relevant for whole and partial brain irradiation. Furthermore, we evaluate the radioprotective potential of resveratrol, a plant polyphenol, which is newly recognized for its bifunctional tumor-preventive and anti-cancer effects. METHODS: Nestin-CFPnuc C57BL/J6 mice, postnatal day 4-6, were decapitated and hippocampal tissue slices, containing the entorhinal-hippocampal formation, cut on a vibratome and cultured on membrane inserts in culture plates. Slices were irradiated on an x-ray machine (0.2-16 Gy) 14-21 days later. Resveratrol (15 µM) was added 2 h before and 24 h after irradiation (IR). BrdU pulse label was conducted for 48 h after IR. Nestin-positive neural stem cells were counted at a confocal life imaging microscope 0, 2, 4, 14, 25, and 42 days after IR. BrdU-positive cells were evaluated by immunofluorescence staining in cryosectioned slices. Cell viability was assessed by CellTiter-Blue fluorometric assay, 3 and 9 days after IR. Dead cells were labelled by propidium iodide (PI). Cytokine concentrations (IL6, KC, MCP-1) were measured in the slice culture medium by cytometric bead array 4 and 24 h after IR. RESULTS: A dose-dependent decline of nestin-positive neural stem cells together with a decrease of proliferation could be detected already at low doses of 0.2 Gy two days after IR. A partial recovery of the stem cell pool was found at late time points (14 and 42 days after IR). PI staining showed a dose-dependent increase of dead cells, reaching significance at 12 Gy. Resveratrol was able to enhance the cell viability significantly in irradiated slices 3 and 9 days after irradiation (4.5 and 8 Gy). IL6, KC and MCP1 release increased 4 and 24 h after IR at 1.5 Gy (IL6) and 3 Gy (KC, MCP1). CONCLUSION: Relatively low IR doses lead to a decrease of nestin-positive stem cells. Our data indicate that this is a result of increased cell death and decreased proliferation but might also be triggered by an increase of inflammation. The neuroprotective action of resveratrol on irradiated hippocampal tissue warrants further investigation.