Mammalian target of rapamycin inhibition protects glioma cells from temozolomide-induced cell death.

Glioblastoma is an incurable brain tumor with a median survival below two years. Trials investigating targeted therapy with inhibitors of the kinase mTOR have produced ambiguous results. Especially combination of mTOR inhibition with standard temozolomide radiochemotherapy has resulted in reduced survival in a phase II clinical trial. To date, this phenomenon is only poorly understood. To recreate the therapeutic setting in vitro, we exposed glioblastoma cell lines to co-treatment with rapamycin and temozolomide and assessed cell viability, DNA damage and reactive oxygen species. Additionally, we employed a novel translatomic based mass spectrometry approach ("mePROD") to analyze acute changes in translated proteins. mTOR inhibition with rapamycin protected glioblastoma cells from temozolomide toxicity. Following co-treatment of temozolomide with rapamycin, an increased translation of reactive oxygen species (ROS)-detoxifying proteins was detected by mass spectrometry. This was accompanied by improved ROS-homeostasis and reduced DNA damage. Additionally, rapamycin induced the expression of the DNA repair enzyme O-6-methylguanine-DNA methyltransferase (MGMT) in glioblastoma cells with an unmethylated MGMT gene promotor. Inhibition of mTOR antagonized the cytotoxic effects of temozolomide in vitro. The induction of antioxidant defences and MGMT are two underlying candidate mechanisms. Further functional experiments in vitro and in vivo are warranted to characterize this effect that appears relevant for combinatorial therapeutic strategies.

Cetuximab-Mediated Protection from Hypoxia- Induced Cell Death: Implications for Therapy Sequence in Colorectal Cancer.

Monoclonal antibodies like cetuximab, targeting the epidermal growth factor receptor (EGFR), and bevacizumab, targeting the vascular endothelial growth factor (VEGF), are an integral part of treatment regimens for metastasized colorectal cancer. However, inhibition of the EGFR has been shown to protect human glioma cells from cell death under hypoxic conditions. In colon carcinoma cells, the consequences of EGFR blockade in hypoxia (e.g., induced by bevacizumab) have not been evaluated yet. LIM1215 and SW948 colon carcinoma and LNT-229 glioblastoma cells were treated with cetuximab, PD153035, and erlotinib and analyzed for cell density and viability. The sequential administration of either cetuximab followed by bevacizumab (CET->BEV) or bevacizumab followed by cetuximab (BEV->CET) was investigated in a LIM1215 (KRAS wildtype) and SW948 (KRAS mutant) xenograft mouse model. In vitro, cetuximab protected from hypoxia. In the LIM1215 model, a survival benefit with cetuximab and bevacizumab monotherapy was observed, but only the sequence CET->BEV showed an additional benefit. This effect was confirmed in the SW948 model. Our observations support the hypothesis that bevacizumab modulates the tumor microenvironment (e.g., by inducing hypoxia) where cetuximab could trigger protective effects when administered later on. The sequence CET->BEV therefore seems to be superior as possible mutual adverse effects are bypassed.

Harmful and nutrient elements in paddy soils and their transfer into rice grains (Oryza sativa) along two river systems in northern and central Vietnam.

Thirty soil samples and 24 corresponding unpolished rice samples along the Red and Huong Rivers in northern and central Vietnam respectively, were analyzed in order to evaluate (a) soil geochemistry, (b) factors that determine the transfer of harmful and nutrient elements from soils into rice grains, (c) health risk to the local population through rice consumption. The concentrations of As, Bi, and U in the soils of this area are higher relative to those of average shale probably due to natural redox-related processes. Also, Zn, Ce, Th, La, Sn, Pb, and Cd are accumulated in some soils because of mining activities or industrial wastewater application. Arsenic concentrations exceed the Vietnamese allowable limit of 15 mg kg-1 in 80% of the tested soils. Twelve percent of the unpolished rice grains surpass the permissible maximum concentration of 0.2 mg Cd kg-1 grain dry matter by FAO/WHO and European Union, and all samples are below the Pb limit. The daily intake of As is within the range of the tolerable intake levels proposed by the European Food Safety Authority. Influences of soil parameters such as pH value, contents of soil organic matter, oxides/hydroxides of Al, Fe, and Mn cause a broad spread of transfer factors from soil to grains. Positive trends exist between the transfer factors within the groups (a) As, Sb, and U, (b) Co, Cu, Ni, and Zn, (c) Cd and Mn which indicate similar influences of soil parameters on their uptake. We propose that the allowable Cd maximum concentration for rice should be set to less than 0.2 mg kg-1. The analysis of As and Cd concentrations in soils and corresponding rice grains as well as the soil pH value should be made obligatory in order to prevent intoxication. In addition, critical elements from nonferrous metal mining and industrial areas should also be evaluated.

Paddy soil geochemistry, uptake of trace elements by rice grains (Oryza sativa) and resulting health risks in the Mekong River Delta, Vietnam.

Soil geochemistry and phytoavailable trace elements were investigated in 80 paddy soil samples and corresponding rice grains from the Mekong River Delta in Vietnam. Soil parameters like Fe-, Al-, and Mn-phases, organic matter, and pH-value determine element concentrations in soil and affect their transfer into rice grains. Arsenic exceeded the allowed limit for Vietnamese agricultural soils in 11% of the samples, presumably caused by natural processes. Lead surpassed the limit in one soil sample. Other toxic elements were close to their natural concentrations and far below allowable limits for agricultural soil. There was no clear correlation of trace element concentrations in soils with those in corresponding grains, even if the different soil parameters and the large pH-range between 3.7 and 6.8 were considered. To assess health risks of critical elements in rice, the thresholds of tolerable upper intake level for total food and drinking water (UL) and of permissible maximum concentration (MC) for rice grains were evaluated. Surprisingly, rice grains grown on non- or low-polluted soils can surpass the upper limits. According to the UL concept, 12% of the grains exceeded the UL of As, 29% that of Cd, and 27% that of Pb for each gender. According to the MC concept, 5% of the rice grains exceeded the MC of inorganic As for adults and 38% that for young children. 24% of the grains surpassed the MC of Pb, while Cd in all grains was below the MC. The differing results of the UL and MC approaches show an urgent need for revision and harmonization concerning As, Cd, and Pb limits, especially regarding countries with high rice consumption.

The physiological mTOR complex 1 inhibitor DDIT4 mediates therapy resistance in glioblastoma.

BACKGROUND: Despite significant advances in the understanding of glioblastoma genetics and biology, survival is still poor. Hypoxia and nutrient depletion in the tumour microenvironment induce adaptive signalling and metabolic responses, which can influence sensitivity to therapeutic regimens. DNA damage-inducible transcript 4 (DDIT4) is a protein induced by hypoxia and in response to DNA stress. Mechanistically, DDIT4 inhibits mammalian target of rapamycin complex 1 (mTORC1) signalling by activation of the tuberous sclerosis 1/2 (TSC1/2) complex. METHODS: Using short hairpin RNA-mediated gene suppression as well as doxycycline-regulated gene induction, we developed a glioblastoma cell model to study effects of DDIT4 under conditions of the glioblastoma microenvironment and therapy. RESULTS: We found an intact DDIT4-mTORC1 signalling axis in human glioblastoma cells that was inducible by hypoxia. Temozolomide and radiotherapy also induced DDIT4 and repressed mTORC1 activity in some glioblastoma cell lines. DDIT4 gene suppression sensitised glioma cells towards hypoxia-induced cell death, while DDIT4 overexpression protected them. Additionally, in clonogenic survival analyses, DDIT4 induction conferred protection from radiotherapy and temozolomide, while DDIT4 gene suppression sensitised cells. CONCLUSIONS: We identified DDIT4 as a cell-intrinsic regulator for adaptive responses and therapy resistance in glioblastoma cells which may interfere with cell death induction by temozolomide, radiotherapy or hypoxia by inhibiting mTORC1 activity.

Disruption of peroxisome proliferator-activated receptor γ coactivator (PGC)-1α reverts key features of the neoplastic phenotype of glioma cells.

The peroxisome proliferator-activated receptor γ coactivator (PGC)-1α is a master regulator of mitochondrial biogenesis and controls metabolism by coordinating transcriptional events. Here, we interrogated whether PGC-1α is involved in tumor growth and the metabolic flexibility of glioblastoma cells. PGC-1α was expressed in a subset of established glioma cell lines and primary glioblastoma cell cultures. Furthermore, a higher PGC-1α expression was associated with an adverse outcome in the TCGA glioblastoma dataset. Suppression of PGC-1α expression by shRNA in the PGC-1α-positive U343MG glioblastoma line suppressed mitochondrial gene expression, reduced mitochondrial membrane potential, and diminished oxygen as well as glucose consumption, and lactate production. Compatible with the known PGC-1α functions in reactive oxygen species (ROS) metabolism, glioblastoma cells deficient in PGC-1α displayed ROS accumulation, had reduced RNA levels of proteins involved in ROS detoxification, and were more susceptible to death induction by H2O2 compared with control cells. PGC-1αsh cells also had impaired proliferation and migration rates in vitro and displayed less stem cell characteristics. Complementary effects were observed in PGC-1α-low LNT-229 cells engineered to overexpress PGC-1α. In an in vivo xenograft experiment, tumors formed by U343MG PGC-1αsh glioblastoma cells grew much slower than control tumors and were less invasive. Interestingly, the PGC-1α knockdown conferred protection against hypoxia-induced cell death, probably as a result of less active anabolic pathways, and this effect was associated with reduced epidermal growth factor expression and mammalian target of rapamycin signaling. In summary, PGC-1α modifies the neoplastic phenotype of glioblastoma cells toward more aggressive behavior and therefore makes PGC-1α a potential target for anti-glioblastoma therapies.

Doxycycline Impairs Mitochondrial Function and Protects Human Glioma Cells from Hypoxia-Induced Cell Death: Implications of Using Tet-Inducible Systems.

Inducible gene expression is an important tool in molecular biology research to study protein function. Most frequently, the antibiotic doxycycline is used for regulation of so-called tetracycline (Tet)-inducible systems. In contrast to stable gene overexpression, these systems allow investigation of acute and reversible effects of cellular protein induction. Recent reports have already called for caution when using Tet-inducible systems as the employed antibiotics can disturb mitochondrial function and alter cellular metabolism by interfering with mitochondrial translation. Reprogramming of energy metabolism has lately been recognized as an important emerging hallmark of cancer and is a central focus of cancer research. Therefore, the scope of this study was to systematically analyze dose-dependent metabolic effects of doxycycline on a panel of glioma cell lines with concomitant monitoring of gene expression from Tet-inducible systems. We report that doxycycline doses commonly used with inducible expression systems (0.01⁻1 µg/mL) substantially alter cellular metabolism: Mitochondrial protein synthesis was inhibited accompanied by reduced oxygen and increased glucose consumption. Furthermore, doxycycline protected human glioma cells from hypoxia-induced cell death. An impairment of cell growth was only detectable with higher doxycycline doses (10 µg/mL). Our findings describe settings where doxycycline exerts effects on eukaryotic cellular metabolism, limiting the employment of Tet-inducible systems.

Alteration of trace element concentrations in plants by adhering particles - Methods of correction.

Trace element concentrations in plants may be influenced by airborne dust or adhering soil particles. Neglecting adhering particles in plant tissue leads to misinterpretation of trace element concentrations in research fields such as phytomining, phytoremediation, bio-monitoring, uptake of micronutrients and provenance studies. In case washing or brushing the samples prior to analysis is insufficient or impossible due to fragile or pre-processed samples mathematical correction should be applied. In this study three methods are presented allowing to subtract the influence of adhering particles in order to obtain the element concentrations in plants resulting only from uptake. All mathematical models are based on trace elements with negligible soil to plant transfer. A prerequisite for the correction methods is trace element analytics with good accuracy and high precision, e.g. through complete acid digestion. In a data set of 1040 plant samples grown in open field and pot trials most plants show a small but detectable amount of adhering particles. While concentrations of nutrients are nearly unaffected trace element concentrations such as Al, Cd, Co, Cr, Fe, Mn, Ni, Pb, REEs, Ti and U may be significantly altered. Different sampling techniques like cutting height can also significantly alter the concentrations measured in the samples.