TTFields alone and in combination with chemotherapeutic agents effectively reduce the viability of MDR cell sub-lines that over-express ABC transporters

<p>Abstract</p> <p>Background</p> <p>Exposure of cancer cells to chemotherapeutic agents may result in reduced sensitivity to structurally unrelated agents, a phenomenon known as multidrug resistance, MDR. The purpose of this study is to investigate cell growth inhibition of wild type and the corresponding MDR cells by Tumor Treating Fields - TTFields, a new cancer treatment modality that is free of systemic toxicity. The TTFields were applied alone and in combination with paclitaxel and doxorubicin.</p> <p>Methods</p> <p>Three pairs of wild type/MDR cell lines, having resistivity resulting from over-expression of ABC transporters, were studied: a clonal derivative (C11) of parental Chinese hamster ovary AA8 cells and their emetine-resistant sub-line Emt^R1; human breast cancer cells MCF-7 and their mitoxantrone-resistant sub lines MCF-7/Mx and human breast cancer cells MDA-MB-231 and their doxorubicin resistant MDA-MB-231/Dox cells. TTFields were applied for 72 hours with and without the chemotherapeutic agents. The numbers of viable cells in the treated cultures and the untreated control groups were determined using the XTT assay. Student t-test was applied to asses the significance of the differences between results obtained for each of the three cell pairs.</p> <p>Results</p> <p>TTFields caused a similar reduction in the number of viable cells of wild type and MDR cells. Treatments by TTFields/drug combinations resulted in a similar increased reduction in cell survival of wild type and MDR cells. TTFields had no effect on intracellular doxorubicin accumulation in both wild type and MDR cells.</p> <p>Conclusions</p> <p>The results indicate that TTFields alone and in combination with paclitaxel and doxorubicin effectively reduce the viability of both wild type and MDR cell sub-lines and thus can potentially be used as an effective treatment of drug resistant tumors.</p

Multilocus ISSR Markers Reveal Two Major Genetic Groups in Spanish and South African Populations of the Grapevine Fungal Pathogen Cadophora luteo-olivacea

Cadophora luteo-olivacea is a lesser-known fungal trunk pathogen of grapevine which has been recently isolated from vines showing decline symptoms in grape growing regions worldwide. In this study, 80 C. luteo-olivacea isolates (65 from Spain and 15 from South Africa) were studied. Inter-simple-sequence repeat-polymerase chain reaction (ISSR-PCR) generated 55 polymorphic loci from four ISSR primers selected from an initial screen of 13 ISSR primers. The ISSR markers revealed 40 multilocus genotypes (MLGs) in the global population. Minimum spanning network analysis showed that the MLGs from South Africa clustered around the most frequent genotype, while the genotypes from Spain were distributed all across the network. Principal component analysis and dendrograms based on genetic distance and bootstrapping identified two highly differentiated genetic clusters in the Spanish and South African C. luteo-olivacea populations, with no intermediate genotypes between these clusters. Movement within the Spanish provinces may have occurred repeatedly given the frequent retrieval of the same genotype in distant locations. The results obtained in this study provide new insights into the population genetic structure of C. luteo-olivacea in Spain and highlights the need to produce healthy and quality planting material in grapevine nurseries to avoid the spread of this fungus throughout different grape growing regions

A dynamic model of the blood-brain barrier "in vitro"

Cell culture models have been widely used for screening of neurotoxicants and represent a viable alternative to direct in vivo experiments. We have developed a dynamic in vitro blood-brain barrier model designed to allow for extensive toxicological, pharmacological and physiological testing. Induction of blood-brain barrier properties in a tri-dimensional hollow fiber culturing apparatus was investigated by co-culturing a bovine aortic endothelial cell line (or rat brain endothelial cells) with rat brain astrocytes (or C6 rat glioma cells) under pulsatile flow conditions to mimic intraluminal blood flow. Cell growth was monitored over time by measuring glucose consumption and lactate production: these experiments confirmed that the hollow fiber cell culturing systems can maintain viable cells in culture for extended (&gt; 1 month) periods of time. Cells were visually inspected after culturing and dissociation from the hollow fiber cartridge and identified as endothelial (by fluorescent Dil-Ac-LDL uptake) or glial (by GFAP immunoreactivity). Blood-brain barrier properties were tested by intraluminal injection of horse-radish peroxidase (HRP, mol. weight 44,000), glucose (m.w. 180) or potassium. Either procedure demonstrated that aortic cells co-cultured with astrocytes (or C6 cells) developed a selective barrier with an estimated electrical resistance of 2,900 omega/cm2. The electrophysiological and morphological properties of BAEC were also affected by the co-culturing process, suggesting that astrocytes induced CNS properties in these cells. These results demonstrate that the hollow fiber cell co-culturing system may be used as a dynamic model of the mammalian blood-brain barrier

Effects of trimethyltin on granule cells excitability in the in vitro rat dentate gyrus

The effects of trimethyltin (TMT) on passive properties and synaptic activity of dentate granule cell (GC) have been investigated in hippocampal slices in vitro. Intracellular recordings from GC indicated that TMT (1 and 10 microM) increased input resistance from 34.1 +/- 3.6 Mohms to 45.6 +/- 4.1 and 64.7 +/- 14.7 Mohms, respectively, 15 min after its application. This was accompanied by a 10-20 mV depolarization. A decrease in IPSP amplitude was also observed, but developed with longer delays (2-4 hr) following TMT exposure. Extracellular recording from the GC layer during paired pulse stimulation of the perforant path showed a decrease in the ratio of the amplitude of the first to the second population spikes (at an interpulse interval of 9 msec), from 1.8 +/- 0.14 to 0.8 +/- 0.08 (p less than 0.05). The amplitude of the first (conditioning) pulse remained unchanged, suggesting that TMT produced a specific decrease of inhibitory efficacy. These results add evidence to the hypothesis that TMT neurotoxicity is mediated by a decrease of inhibitory synaptic functions

Hyperpolarization-activated ion currents in cultured rat cortical and spinal cord astrocytes

Hyperpolarization-activated currents were recorded f r om r a t b r a i n c o r t i cal and spinal cord astrocytes maintained i n culture. Spinal cord astrocytes expressed pr i m a r i l y an i n w a r d rectifier potassium current characterized by time-dependent inactivation, a strong dependence on extracellular N a + and insensitivity to i n t r a c e l l u l a r GTP- -y-S ( 0 . 2 mM). I n cortical astrocytes voltage clamp protocols aimed to elicit currents activated at, or negative to cell membrane potentials led to the development of two distinct ion currents. The most prominent current resembled the inwar d rectifier potassium current. This component was sensitive to blockade by extracellular cesium and was greatly reduced d u r i n g recordings performed w i t h GTP-7-S ( 0 . 2 Mm) added to the pipette solutions. The remaining current component was similar to the endothelial I h a current. I h a conductance was enhanced by extracellular potassium and the current reversal potential behaved as expected for a mixed cation, N a 7 K + current. I h a was nearly abolished after removal of extracellular Na". These results are consistent w i t h the expression of a novel mixed cation conductance i n g l i a l cells, possibly involved i n extracell u l a r potassium bufferin

Gangliosides' dual mode of action: A Working hypothesis

Using in vitro preparations, we have tested the hypothesis that gangliosides, and more specifically GM1, may prevent progressive neural damage following a trauma by means of complex intracellular mechanisms that might be triggered originally by ganglioside interaction with neuronal membranes. We have recently shown that 2-hr ganglioside incubation in vitro stimulates the membrane Na/K pump in neuromuscular preparations. However, 5-6 hr incubation or in vivo treatment for 3 days with a daily injection of gangliosides at a dose of 1 or 10 mg/kg prevents the depolarization that normally occurs after several hours of exposure to K+-free solutions. In such undepolarized muscles, the electrogenic Na+/K+ pump does not seem to be activated. Hippocampal slices subjected to hypoxia undergo depolarization, which is reversed after oxygen readmission. The recovery phase is characterized by a huge hyperpolarization, probably reflecting electrogenic pump activity. In control preparations the depolarization occurs after 3.15 \ub1 0.4 min and has a value of 48.7 \ub1 5.7 Mv; GM1 treatment for at least 4-5 hr increases the latency to 7.3 \ub1 2.3 min, and the depolarization is reduced to 31.8 \ub1 4.5 mV. This protective effect is accompanied by a reduced hyperpolarization in treated preparations. The ionic studies performed on neuromuscular preparations indicate that the protective effect may be not solely dependent on K+ leakage; however, the experiments are not conclusive and must be repeated with more direct methods. The results obtained indicate a dual mode of action for gangliosides. The early one seems characterized by membrane-enzyme activation, perhaps in relationship to their incorporation in the membrane, which could be compatible with previously described effects, such as enhancement of neuronal sprouting and neuritogenesis. The late one, occurring 4-5 hr after ganglioside addition in vitro, might reflect intracellular events and be compatible with the protective action exhibited by gangliosides against neural damage

INVIVO TREATMENT WITH GM1 PREVENTS THE RAPID DECAY OF ATPASE ACTIVITIES AND MITOCHONDRIAL DAMAGE IN HIPPOCAMPAL SLICES

Slices from rat CA3 hippocampal area show a 30% decrement in ATPase activity after 35 min of 'in vitro' incubation. Such a drop is accompanied by an alteration of mitochondrial ultrastructure. However, if rats are treated daily with GM1 ganglioside (10 mg/kg during 3 days) both phenomena are fully prevented. These results would suggest a protective effect of gangliosides onto membrane structures under stress conditions