Induction of Mitosis Delay And Apoptosis By RTA 404 In Glioblastoma Multiforme

Background Glioblastoma multiforme (GBM) is the vicious malignant brain tumor in adults. Despite advances multi-disciplinary treatment, GBM constinues to have a poor overall survival.CDDO-trifluoroethyl-amide, a trifluoroethylamidederivative of CDDO, is an Nrf2/ARE pathway activator. RTA 404 is used to inhibit proliferation and induce differentiation and apoptosis in glioma cells. However, it not clear what effect it may have on tumorigenesis in GBM.MethodsThis in vitro study evaluated the effects of RTA 404 on GBM cells. To do this, we treated GBM8401 cell lines with RTA 404 and assessed apoptosis, cell cycle. DNA content and induction of apoptosis were analyzed by flow cytometry and protein expression by Western blot analysis.ResultsRTA 404 significantly inhibited the proliferation induced cell apoptosis on GBM 8401 cell line. Typical plasma membrane undergoes structural changes that cause translocation of phosphatidylserine from the inside to outside. Due to cell external pressure cause mitochondrial membrane potential change lead to cell apoptosis. Caspase-3 active respond to apoptosis phenomenon, continuous progression of apoptosis.In addition, treatment with RTA 404 led to an accumulation of G2/M-phase cells. An analysis of Cyclin B1, CDK1 and Cyclin B1/CDK1 complex association suggested that cell cycle progression seems also to be regulated by RTA 404. Therefore, RTA 404 may not only induced cell cycle G2/M arrest, it may also exert apoptosis in established GBM cells. ConclusionRTA 404 can inhibit proliferation, cell cycle progression and induce apoptosis in GBM cells in vitro, possibly though its inhibition of Cyclin B1, CDK1 expression, and Cyclin B1/CDK1 association.

A Sonosensitizer-based Polymeric Nanoplatform for Realizing Chemo-sonodynamic Combination Therapy of Lung Cancer

BackgroundLung cancer was regarded as the most common type of tumors worldwide, and the relative lethality was considerably high. However, since the tumor tissues located in deeper parts in human bodies, the traditional technologies like photodynamic therapy could not reached desired effect. Sonosensitizers can induce sonodynamic therapy, possessing the benefits of deep penetration and effective tumor inhibition by generating reactive oxygen. Besides, ultrasound possessed the ability of deep penetration, which showed favorable tumor inhibition effect.ResultsA redox/enzyme/ultrasound responsive Rhein-chondroitin sulfate nano-preparation encapsulating docetaxel was initially fabricated. The nanoparticles show increased cellular uptake with quick drug release, revealing good stability and monodispersed form in physiological environment. Also, Rhein could induce apoptosis and mitochondrial membrane potential change, enhancing the expression of apoptosis-related protein. Further, the inhibited metastasis and angiogenesis of cancer cells by sonodynamic therapy would lead to reduced expression of M2 type macrophages.ConclusionRhein was firstly discovered to possess the sonodynamic effect in this work by generating plenty of reactive oxygen after treating by ultrasound. The nanoplatform enhanced synergistic anti-tumor effect by both sonodynamic therapy and chemotherapeutic efficacy, and showed benign biocompatibility, which might pave a way for the investigation of chemo-sonodynamic therapy with different cancers.

Real-time detection of changes in yeast plasma membrane potential using genetically encoded voltage indicator proteins

ABSTRACT In yeast, adaptation to varying conditions often requires proper regulation of the plasma membrane potential. To determine yeast membrane potential change, optical methods involving potentiometric dyes have been supplemental to the direct electrode-based method. However, the hydrophobic nature of the dyes and their slow distribution across the membrane still limits their utilization. Genetically encoded voltage indicator (GEVI) proteins employed in neuroscience offer a tantalizing alternative for monitoring yeast membrane potential change. In this work, several widely used GEVI proteins were assessed in Saccharomyces cerevisiae for their expression and function as a voltage reporter. Among them, only ArcLight and Accelerated Sensor of Action Potential (ASAP) proteins could be expressed and transported to the plasma membrane. While the voltage-sensing capability was demonstrated for both ArcLight and ASAP, ArcLight fluorescence was sensitive to the intracellular pH change concurrently with the voltage change. Therefore, we established that ASAP is the more suitable GEVI protein for reporting yeast membrane potential change. This voltage-sensing reporter for yeast based on ASAP offers a new effective strategy for real-time optical detection of yeast membrane potential change, which potentially facilitates many areas of yeast research including optimizing growth conditions for industrial use and investigating yeast ion transport system.

Damaging Effects of Bisphenol A on the Kidney and the Protection by Melatonin: Emerging Evidences from In Vivo and In Vitro Studies

This study investigates the effects of bisphenol A (BPA) contamination on the kidney and the possible protection by melatonin in experimental rats and isolated mitochondrial models. Rats exposed to BPA (50, 100, and 150 mg/kg, i.p.) for 5 weeks demonstrated renal damages as evident by increased serum urea and creatinine and decreased creatinine clearance, together with the presence of proteinuria and glomerular injuries in a dose-dependent manner. These changes were associated with increased lipid peroxidation and decreased antioxidant glutathione and superoxide dismutase. Mitochondrial dysfunction was also evident as indicated by increased reactive oxygen species production, decreased membrane potential change, and mitochondrial swelling. Coadministration of melatonin resulted in the reversal of all the changes caused by BPA. Studies using isolated mitochondria showed that BPA incubation produced dose-dependent impairment in mitochondrial function. Preincubation with melatonin was able to sustain mitochondrial function and architecture and decreases oxidative stress upon exposure to BPA. The findings indicated that BPA is capable of acting directly on the kidney mitochondria, causing mitochondrial oxidative stress, dysfunction, and subsequently, leading to whole organ damage. Emerging evidence further suggests the protective benefits of melatonin against BPA nephrotoxicity, which may be mediated, in part, by its ability to diminish oxidative stress and maintain redox equilibrium within the mitochondria.

Current injection and receptor-mediated excitation produce similar maximal firing rates in hypoglossal motoneurons

The maximum firing rates of motoneurons (MNs), activated in response to synaptic drive, appear to be much lower than that elicited by current injection. It could be that the decrease in input resistance associated with increased synaptic activity (but not current injection) might blunt overall changes in membrane depolarization and thereby limit spike-frequency output. To test this idea, we recorded, in the same cells, maximal firing responses to current injection and to synaptic activation. We prepared 300 μm medullary slices in neonatal rats that contained hypoglossal MNs and used whole-cell patch-clamp electrophysiology to record their maximum firing rates in response to triangular-ramp current injections and to glutamate receptor-mediated excitation. Brief pressure pulses of high-concentration glutamate led to significant depolarization, high firing rates, and temporary cessation of spiking due to spike inactivation. In the same cells, we applied current clamp protocols that approximated the time course of membrane potential change associated with glutamate application and with peak current levels large enough to cause spike inactivation. Means (SD) of maximum firing rates obtained in response to glutamate application were nearly identical to those obtained in response to ramp current injection [glutamate 47.1 ± 12.0 impulses (imp)/s, current injection 47.5 ± 11.2 imp/s], even though input resistance was 40% less during glutamate application compared with current injection. Therefore, these data suggest that the reduction in input resistance associated with receptor-mediated excitation does not, by itself, limit the maximal firing rate responses in MNs.

The Apoptotic Activity of one VLC Fraction of the Sponge Petrosia tuberosa on Human Cervical Cells and the Subsequent Isolation of a Bioactive Polyacetylene

As part of our ongoing studies on bioactive natural products from marine sponges, we investigated the cytotoxic potential of extracts from the new sponge Petrosia tuberosa sampled from Mauritius waters. Bioguided fractionation of the ethyl acetate extract by vacuum liquid chromatography (VLC) revealed two fractions, namely VLC (6-9) and (13-17) showing cell deaths of 86 ± 1% and 88 ± 4%, respectively, at 50 μg/mL on HeLa cells. At 10 μg/mL, only VLC (13-17) displayed a significant cell death (56 ± 7%) compared with VLC (6-9) (8 ± 1 %). The cytotoxic activity of VLC (13-17) was also determined on nine other human cancer cell lines. Clonogenic assay, mitochondrial membrane potential change, DNA fragmentation and microscopic analysis of fraction VLC (13-17) revealed distinct features of apoptosis on HeLa cells. Further fractionation and purification of this fraction by chromatographic techniques resulted in isolation of one known secondary metabolite, petrosynol. Its structure was determined by 1H and 13C-NMR analyses.

Alternative NADH dehydrogenase (NDH2): intermembrane-space-facing counterpart of mitochondrial complex I in the procyclic Trypanosoma brucei

SUMMARYThe respiratory chain of the procyclic stage of Trypanosoma brucei contains the standard complexes I through IV, as well as several alternative enzymes contributing to electron flow. In this work, we studied the function of an alternative NADH : ubiquinone oxidoreductase (NDH2). Depletion of target mRNA was achieved using RNA interference (RNAi). In the non-induced and RNAi-induced cell growth, membrane potential change, alteration in production of reactive oxygen species, overall respiration, enzymatic activities of complexes I, III and/or IV and distribution of NADH : ubiquinone oxidoreductase activities in glycerol gradient fractions were measured. Finally, respiration using different substrates was tested on digitonin-permeabilized cells. The induced RNAi cell line exhibited slower growth, decreased mitochondrial membrane potential and lower sensitivity of respiration to inhibitors. Mitochondrial glycerol-3-phosphate dehydrogenase was the only enzymatic activity that has significantly changed in the interfered cells. This elevation as well as a decrease of respiration using NADH was confirmed on digitonin-permeabilized cells. The data presented here together with previously published findings on complex I led us to propose that NDH2 is the major NADH : ubiquinone oxidoreductase responsible for cytosolic and not for mitochondrial NAD+ regeneration in the mitochondrion of procyclic T. brucei.

CaMKII inhibition hyperpolarizes membrane and blocks nitrergic IJP by closing a Cl− conductance in intestinal smooth muscle

The ionic basis of nitrergic “slow'” inhibitory junction potential (sIJP) is not fully understood. The purpose of the present study was to determine the nature and the role of calmodulin-dependent protein kinase II (CaMKII)-dependent ion conductance in nitrergic neurotransmission at the intestinal smooth muscle neuromuscular junction. Studies were performed in guinea pig ileum. The modified Tomita bath technique was used to induce passive hyperpolarizing electrotonic potentials (ETP) and membrane potential change due to sIJP or drug treatment in the same cell. Changes in membrane potential and ETP were recorded in the same smooth muscle cell, using sharp microelectrode. Nitrergic IJP was elicited by electrical field stimulation in nonadrenergic, noncholinergic conditions and chemical block of purinergic IJP. Modification of ETP during hyperpolarization reflected active conductance change in the smooth muscle. Nitrergic IJP was associated with decreased membrane conductance. The CAMKII inhibitor KN93 but not KN92, the Cl− channel blocker niflumic acid (NFA), and the KATP-channel opener cromakalim hyperpolarized the membrane. However, KN93 and NFA were associated with decreased and cromakalim was associated with increased membrane conductance. After maximal NFA-induced hyperpolarization, hyperpolarization associated with KN93 or sIJP was not seen, suggesting a saturation block of the Cl− channel signaling. These studies suggest that inhibition of CaMKII-dependent Cl− conductance mediates nitrergic sIJP by causing maximal closure of the Cl− conductance.