Glucose starvation impairs DNA repair in tumour cells selectively by blocking histone acetylation.

BACKGROUND AND PURPOSE: Tumour cells are characterized by aerobic glycolysis and thus have high glucose consumption. Because repairing radiation-induced DNA damage is an energy-demanding process, we hypothesized that glucose starvation combined with radiotherapy could be an effective strategy to selectively target tumour cells. MATERIAL AND METHODS: We glucose-starved tumour cells (A549, FaDu) in vitro and analysed their radiation-induced cell responses compared to normal fibroblasts (HSF7). RESULTS: Irradiation depleted intracellular ATP levels preferentially in cancer cells. Consequently, glucose starvation impaired DNA double-strand break (DSB) repair and radiosensitized confluent tumour cells but not normal fibroblasts. In proliferating tumour cells glucose starvation resulted in a reduction of proliferation, but failed to radiosensitize cells. Glucose supply was indispensable during the late DSB repair in confluent tumour cells starting approximately 13 h after irradiation, and glucose starvation inhibited radiation-induced histone acetylation, which is essential for chromatin relaxation. Sirtinol - an inhibitor of histone deacetylases - reverted the effects of glucose depletion on histone acetylation and DNA DSB repair in tumour cells. Furthermore, a glucose concentration of 2.8 mmol/L was sufficient to impair DSB repair in tumour cells and reduced their clonogenic survival under a fractionated irradiation regimen. CONCLUSIONS: In resting tumour cells, glucose starvation combined with irradiation resulted in the impairment of late DSB repair and the reduction of clonogenic survival, which was associated with disrupted radiation-induced histone acetylation. However, in normal cells, DNA repair and radiosensitivity were not affected by glucose depletion.

Inhibition of colonic tumor growth by the selective SGK inhibitor EMD638683.

BACKGROUND: The serum and glucocorticoid inducible kinase SGK1, which was originally cloned from mammary tumor cells, is highly expressed in some but not all tumors. SGK1 confers survival to several tumor cells. Along those lines, the number of colonic tumors following chemical carcinogenesis was decreased in SGK1 knockout mice. Recently, a highly selective SGK inhibitor (EMD638683) has been developed. The present study explored whether EMD638683 affects survival of colon carcinoma cells in vitro and impacts on development of colonic tumors in vivo. METHODS: Colon carcinoma (Caco-2) cells were exposed to EMD638683 with or without exposure to radiation (3 Gray) and cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin V binding, mitochondrial potential from JC-9 fluorescence, caspase 3 activity from CaspGlow Fluorescein staining, DNA degradation from propidium iodide staining as well as late apoptosis from annexin-V FITC and propidium iodide double staining. In vivo tumor growth was determined in wild type mice subjected to chemical carcinogenesis (intraperitoneal injection of 20 mg/kg 1,2-dimethylhydrazine followed by three cycles of 30 g/L synthetic dextran sulfate sodium in drinking water for 7 days). RESULTS: EMD638683 treatment significantly augmented the radiation-induced decrease of forward scatter, increase of phosphatidylserine exposure, decrease of mitochondrial potential, increase of caspase 3 activity, increase of DNA fragmentation and increase of late apoptosis. The in vivo development of tumors following chemical carcinogenesis was significantly blunted by treatment with EMD638683. CONCLUSIONS: EMD638683 promotes radiation-induced suicidal death of colon tumor cells in vitro and decreases the number of colonic tumors following chemical carcinogenesis in vivo.

EGFR-mediated stimulation of sodium/glucose cotransport promotes survival of irradiated human A549 lung adenocarcinoma cells.

BACKGROUND AND PURPOSE: Solid tumor cells may adapt to an ischemic microenvironment by upregulation of sodium/glucose cotransport (SGLT) in the plasma membrane which supplies the tumor cell with glucose even at very low extracellular glucose concentration. Since SGLT activity has been shown to depend on the epithelial growth factor receptor (EGFR) and EGFR reportedly is activated by ionizing radiation, we tested for irradiation-induced SGLT activity. MATERIALS AND METHODS: A549 lung adenocarcinoma and FaDu head and neck squamous cancer cells were irradiated with 0 and 4 Gy X-ray and electrogenic SGLT transport activity was recorded by patch clamp current clamp in the presence and absence of extracellular glucose (5mM), the SGLT inhibitor phlorizin (500 µM), and the inhibitor of the EGFR tyrosine kinase activity erlotinib (1 µM). In addition, the effect of phlorizin and erlotinib on glucose uptake and clonogenic survival was tested in irradiated and control cells by tracer flux and colony formation assays, respectively. RESULTS: Irradiated A549 cells exhibited a significantly lower membrane potential 3h after irradiation than the control cells. Phlorizin, erlotinib or removal of extracellular glucose, hyperpolarized the irradiated A549 cells to a significantly higher extent than the control cells. Similarly, but less pronounced, glucose removal hyperpolarized irradiated FaDu cells. In addition, irradiated A549 cells exhibited a highly increased (3)H-glucose uptake which was sensitive to phlorizin. Finally, phlorizin radiosensitized the A549 and FaDu cells as evident from the colony formation assays. CONCLUSIONS: Taken together, these data suggest an irradiation-stimulated and EGFR-mediated increase in SGLT-generated glucose uptake which is required for the survival of the genotoxically stressed tumor cells.

Inhibition of cyclooxygenase-2 activity by celecoxib does not lead to radiosensitization of human prostate cancer cells in vitro.

PURPOSE: To evaluate the potential radiosensitizing effect of the specific COX-2 inhibitor celecoxib (Celebrex) on prostate carcinoma cells in vitro. MATERIALS AND METHODS: The influence of celecoxib (concentration range 5 to 75 microM) on radiation-induced cellular and clonogenic survival was investigated in prostate carcinoma cell lines PC-3, DU145, LNCaP and normal prostate epithelial cells (PrEC). Western blot analysis and ELISA were used to determine the impact of radiation alone or radiation combined with celecoxib treatment on COX-2 expression and prostaglandin E2 synthesis. To evaluate induction of celecoxib-induced apoptosis cell cycle analysis has been performed. RESULTS: Celecoxib (5, 10 and 25 microM) in combination with single-dose irradiation of 2 Gy induced a significant radiosensitization in normal prostate epithelial cells which could not be observed for any of the prostate carcinoma cell lines investigated. Increased COX-2 protein expression in PC-3 cells was obvious only after IR with 15 Gy, while PGE2 production was elevated following irradiation (2-15 Gy) in a dose-dependent manner. Treatment with celecoxib alone or in combination with IR led to a dose-dependent increase in COX-2 protein expression. Nevertheless pre-treatment with celecoxib caused a marked reduction of radiation-induced enzyme activity as tested at the level of PGE2 production, both in PC-3 and DU145 cells. Following fractionated irradiation with single doses of 2 Gy, elevated COX-2 protein expression as well as enhanced PGE2 production was observed already after the second fraction in PC-3 cells. Pre-treatment with celecoxib reduced the amount of PGE(2) significantly, but not of COX-2 protein. CONCLUSIONS: Our data obtained for the human prostate cancer cell lines do not indicate that a marked inhibition of prostaglandin E2 synthesis by celecoxib leads to enhanced radiosensitization. Thus, in terms of radiosensitization the analysed prostate cancer cells can be classified as non-responders to celecoxib treatment.

Inhibition of radiation-induced EGFR nuclear import by C225 (Cetuximab) suppresses DNA-PK activity.

BACKGROUND AND PURPOSE: Inhibition of EGFR-function can induce radiosensitization in tumor cells. Purpose of our investigation was to identify the possible molecular mechanism of radiosensitization following treatment with anti-EGFR-antibody C225 (Cetuximab). MATERIALS AND METHODS: The effect of C225 on radiation response was determined in human cell lines of bronchial carcinoma (A549) and breast adenoma cells (MDA MB 231). The molecular effects of C225 on EGFR-function after irradiation were analyzed applying western blotting, immune-precipitation and kinase assays. Effects on DNA-repair were detected by quantification of gamma-H2AX positive foci 24h after irradiation. RESULTS: The EGFR specific antibody C225 induced radiosensitization in A549 and also in MDA MB 231 cells. Radiosensitization in A549 was associated with blockage of radiation-induced EGFR transport into the nucleus, and immobilized the complex of EGFR with DNA-dependent protein kinase (DNA-PK) in the cytoplasm. As a consequence radiation-induced DNA-PK activation was abolished, a process that is essential for DNA-repair after radiation exposure. Likewise C225 treatment increased the residual amount of gamma-H2AX-positive foci 24h after irradiation in A549 and in MDA MB 231 cells. CONCLUSIONS: Our results suggest that irradiation induced DNA-PK activation-essential for DNA repair-may be hampered specifically by use of the anti-EGFR-antibody C225. This process is associated with radiosensitization.

Influence of growth factors on the proliferation of vascular smooth muscle cells isolated from subtotally nephrectomized rats after endothelin or angiotensin II antagonism.

BACKGROUND: Cardiovascular disease is the most important cause of death in patients with end-stage renal disease. In uraemia, the renin-angiotensin-aldosterone and endothelin (ET) systems are activated. It is not known whether inhibition of these systems attenuates the proliferation of isolated smooth muscle cells of uraemic rats. METHODS: Subtotally nephrectomized (SNX) rats were treated with an ET(A) receptor antagonist, an ET(AB) receptor antagonist, the angiotensin type 1 (AT1) receptor antagonist losartan (all 10 mg/kg body weight/day) or the angiotensin-converting enzyme (ACE) inhibitor trandolapril (0.1 mg/kg body weight/day) or received no medication (SNX) for 12 weeks. Then, aortal smooth muscle cells (SMCs) were isolated and cultivated. After incubation of SMCs with different growth factors (5-7 days), proliferation was measured using a bromodeoxyuridine enzyme-linked immunosorbent assay (BrdU ELISA). RESULTS: Higher maximum levels of proliferation were found in SMCs from untreated SNX rats than in SMCs from control animals [platelet-derived growth factor-BB (PDGF-BB) 486.60+/-8.27 vs 346.74+/-4.60%, basic fibroblast growth factor (bFGF) 176.68+/-6.50 vs 123.71+/-1.49%, tumour necrosis factor-alpha (TNF-alpha) 153.38+/-10.16 vs 122.27+/-1.41%]. Treatment with ET receptor antagonists or losartan attenuated growth factor-stimulated proliferation (PDGF-BB: ET(A) receptor antagonist, 135.71+/-1.08%; ET(AB) receptor antagonist, 122.72+/-0.58%; losartan: 103.69+/-1.83%, n = 8). SMCs from trandolapril-treated rats showed an increased response (PDGF-BB 663.48+/-7.00%, n = 8). CONCLUSIONS: Treatment of SNX rats with ET receptor antagonists or losartan reduced growth factor-induced SMC proliferation in vitro. However, further investigations with uraemic patients have to clarify whether angiotensin or ET receptor antagonists inhibit the development of atherosclerosis.

Molecular targeting in radiotherapy of lung cancer.

Molecular targeting is a promising option to increase the radiation response of tumours and to decrease normal tissue reactions, i.e. to achieve therapeutic gain. Molecular targeting substances in themselves are not curative while radiation is a highly efficient cytotoxic agent, with local recurrences often occurring from only few surviving clonogenic cells. High-dose radiotherapy therefore offers optimal conditions to evaluate the potential of specific biology-driven drugs for oncology. This review summarises the current status of preclinical and clinical research on combined radiation with examples of molecular targeting substances relevant for the treatment of NSCLC (EGFR, COX-2, VEGFR, KGF, TGF-beta, BBI).

Biological aspects of radiation and drug-eluting stents for the prevention of restenosis.

Based on recent advances, this article aims to review the biological basis for the use of either radiation or drug-eluting stents for the prevention of restenosis, and to elucidate the complementary role that they may play in the future. Vascular restenosis is a multifactorial process primarily driven by the remodeling of the arterial wall, as well as by the hyperproliferation of smooth muscle cells (SMC). These pathophysiological features are the target of therapeutic strategies aimed at inhibiting constrictive remodeling as well as inhibiting SMC proliferation. The success of radiation as well as anti-proliferative drugs such as paclitaxel and sirolimus lies in the primary and/or multifactorial inhibition of cell proliferation. Radiation has the additional feature of preventing constrictive remodeling while sirolimus has the potential property of being anti-inflammatory, which may be a desirable feature. The effects of radiation are not reliant on any uptake and "metabolism" by the target cells, as in the case with drugs, and thus radiation potentially may be more effective as a result of its more-direct action. However, radiation does have some significant drawbacks compared to drug-eluting stents, including a much delayed re-endothelialization resulting in the need for prolonged anti-platelet therapy. Based on recent clinical data, drug-eluting stents have been shown to markedly reduce the likelihood of restenosis, which actually favors this approach for the prevention of restenosis. From a biological perspective, drug-eluting stents and radiation have certain differences, which are reviewed in this article.

BCL-2 family proteins modulate radiosensitivity in human malignant glioma cells.

Radiotherapy is the standard treatment for glioblastoma. Here, we assessed the radiosensitivity of 12 human malignant glioma cell lines in vitro and correlated these data with irradiation-induced cell cycle changes, chemosensitivity profiles and BCL-2 family protein expression. Irradiation at 3 Gy failed to cause major cell cycle perturbations. Radioresistance was associated with collateral sensitivity to the topoisomerase II inhibitors, teniposide and doxorubicin. High levels of BCL-XL and low levels of BAX were independently linked to radioresistance. Ectopic expression of a BAX transgene induced radiosensitization in the LN-18 cell line. Thus, BCL-2 family protein expression modulates radiosensitivity in human glioma cells and targeted alterations in BCL-2 family protein expression are a promising strategy to improve the therapeutic efficacy of radiotherapy for gliomas.