Changes In Transmembrane Potential Research Articles

Butein Induces Apoptosis in Human Uveal Melanoma Cells Through Mitochondrial Apoptosis Pathway

Purpose: To study the cytotoxic effects and related signaling pathways of butein on human uveal melanoma cells in vitro.Materials and Methods: Three human uveal melanoma cell lines (M17, SP6.5, and C918), retinal pigment epithelial (RPE) cells and scleral fibroblasts were treated with butein at different dosages. The effects of butein on cell viability were assessed by using the MTT assay. Cell apoptosis was determined using annexin V-FITC/ethidium homodimer III flow cytometry. Mitochondrial transmembrane potential changes were assessed by using the JC-1 fluorescent reader, cytosol cytochrome c levels, and the activities of caspase-3, -8, and -9 were measured by using an enzyme-linked immunosorbent assay or colorimetric assay.Results: Butein reduced the cell viability of cultured human uveal melanoma cells in a dose-dependent manner (10, 30, and 100 μM), with IC50 at 13.3 μM and 15.8 μM in SP6.5 and M17 cell lines, respectively. Similar effects were also found in a highly aggressive and metastatic C918 cell line (IC50 16.7 μM). Butein at lower concentrations (10–30 μM) selectively reduced the cell viability of uveal melanoma cells, without affecting cell viability of RPE cells and fibroblasts. Butein-induced apoptosis of melanoma cells, increased mitochondrial permeability and the level of cytosol cytochrome c, caspase-9 and -3 activities (but not caspase-8) in a dose-dependent manner.Conclusions: Butein has selectively potent pro-apoptotic effects on cultured human uveal melanoma cells via the intrinsic mitochondrial pathway.

Effect of tilorone and its analogues on the change of mitochondrial potential of rat hepatocytes

The influence of tilorone dihydrochloride and its analogues--diphenyl derivatives on the changes of transmembrane potential of mitochondrial membranes of the isolated rat hepatocytes has been estimated. Authors have shown a significant increase in mitochondrial potential thirty minutes after the introduction of the test compounds to the cells using the fluorescent probe JC-1. These results indicate the rapid activation with tilorone and its analog--dihydrochloryde 4,4'-bis-[2-(diethylamino)ethoxy]diphenyl--of the RLR signaling pathway. The final stage of this pathway is the cell production of IFN type I. The authors concluded that there is an increasing of the organelles resistance to the extra/intracellular damaging agents under the influence of the test compounds.

Abstract 2001: Caffeic acid phenethyl ester inhibits growth of human lung adenocarcinoma cells with an epidermal growth factor receptor T790M mutation susceptible property

Abstract Purpose: Target therapeutics targeting epidermal growth factor receptor (EGFR) effectively kills non-small cell lung cancer cells along with a high proportion acquires T790M mutation of EGFR, an amino acid alteration likely causing drug resistance. Caffeic acid phenethyl ester (CAPE), a naturally occurring small molecule isolated from honeybee propolis, is known to possess preferential bioactivity against lung cancer cells. Materials and Methods: In this study, we evaluated the effect of CAPE on A549 (EGFR wild-type) and H1975 (EGFR T790M mutation) human lung adenocarcinoma cells by using assays as follows: MTS for viability, DNA histogram for hypoploidy and cell cycle analysis, DiOC6(3) staining for mitochondrial transmembrane potential, and Western blotting for caspase-3 cleavage Results: We found that CAPE inhibited the growth of lung adenocarcinoma H1975 cells with an extent greater than A549 (IC50 approximate 5 versus 10 mg/mL) after 24 - 48 h treatment. This differential growth inhibition was accompanied by much higher percentages of sub-G1 (7.5 folds) and G2/M arrest (2.6 folds) in H1975 cells. No significant difference could be seen for changes in mitochondrial transmembrane potential and caspase-3 cleavage, indicating a mitochondria-independent and caspase-3-inependent pathway involved. Conclusion: It suggests that CAPE may have potential to be developed as a small-molecule therapeutics against lung adenocarcinoma cells harboring wild-type epidermal growth factor receptor and, more effectively, the T790M-mutated clone. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2001. doi:1538-7445.AM2012-2001

TDCS possibly stimulates glial cells

ObjectiveExplore the possibility that transcranial direct current stimulation (tDCS) of the brain affects glial cells. MethodsCable theory is used to estimate roughly transmembrane potential in neurons and glial cells. tDCS is additionally compared to neuronal stimulation techniques for which the mechanisms are well known. ResultsTheoretical calculations indicated that tDCS can affect the glial transmembrane potential. The change is similar to what is physiologically observed in astrocytes during neuronal activation. In neurons, transmembrane potential changes are much weaker than the threshold for eliciting action potentials. ConclusionsBased on simplified cable theory, tDCS may affect glial cells’ transmembrane potential and thereby the balance of neurotransmitters. No physiological evidence or proof is available, however. SignificanceIt is an exciting possibility that tDCS could manipulate glial cells because they are active participants in brain function, and have multiple essential roles in the human brain. This approach may change greatly the therapeutic potential of tDCS, and also affects the safety considerations.

Oxidative stress induced by crude venom from the jellyfish Pelagia noctiluca in neuronal-like differentiated SH-SY5Y cells

Marine toxins are a suitable research model and their mechanism of action is intriguing and still under debate. Either a pore formation mechanism or oxidative stress phenomena may explain the damage induced by toxins. The effect of crude venom from isolated nematocysts of the jellyfish Pelagia noctiluca on neuronal-like cells derived from human neuroblastoma SH-SY5Y has been here studied. To prove the possible oxidative stress events, cell viability, assessed by MTT quantitative colorimetric assay, intracellular reactive oxygen species (ROS) quantified by the non-fluorescent probe H2DCF-DA and changes in mitochondrial transmembrane potential (ΔΨm) measured by the incorporation of a cationic fluorescent dye rhodamine-123 were verified on venom-treated cells (0.05–0.5μg/ml doses). A dose- and time-dependent reduction of all parameters was observed after venom treatment. NAC (N-acetyl-cysteine), antioxidant applied before crude venom application, significantly counteracted the decrease in cell viability and ROS production, while ΔΨm was only partially restored. The disruption of mitochondrial membrane by P. noctiluca crude venom may thus induce oxidative stress by inhibiting mitochondrial respiration and uncoupling oxidative phosphorylation, sensitizing mitochondria in SH-SY5H cells and facilitating membrane permeability. In sum, our findings suggest that P. noctiluca crude venom directly induces ΔΨm collapse with further generation of ROS and add novel information to the understanding of such toxins, still not completely clarified.

Coupling between the voltage-sensing and pore domains in a voltage-gated potassium channel

Voltage-dependent potassium (Kv), sodium (Nav), and calcium channels open and close in response to changes in transmembrane (TM) potential, thus regulating cell excitability by controlling ion flow across the membrane. An outstanding question concerning voltage gating is how voltage-induced conformational changes of the channel voltage-sensing domains (VSDs) are coupled through the S4–S5 interfacial linking helices to the opening and closing of the pore domain (PD). To investigate the coupling between the VSDs and the PD, we generated a closed Kv channel configuration from Aeropyrum pernix (KvAP) using atomistic simulations with experiment-based restraints on the VSDs. Full closure of the channel required, in addition to the experimentally determined TM displacement, that the VSDs be displaced both inwardly and laterally around the PD. This twisting motion generates a tight hydrophobic interface between the S4–S5 linkers and the C-terminal ends of the pore domain S6 helices in agreement with available experimental evidence.

Intense picosecond pulsed electric fields induce apoptosis through a mitochondrial-mediated pathway in HeLa cells

The application of pulsed electric fields (PEF) is emerging as a new technique for tumor therapy. Picosecond pulsed electric fields (psPEF) can be transferred to target deep tissue non-invasively and precisely, but the research of the biological effects of psPEF on cells is limited. Electric theory predicts that intense psPEF will target mitochondria and lead to changes in transmembrane potential, therefore, it is hypothesized that it can induce mitochondrial-mediated apoptosis. HeLa cells were exposed to psPEF in this study to investigate this hypothesis. MTT assay demonstrated that intense psPEF significantly inhibited the proliferation of HeLa cells in a dose-dependent manner. Typical characteristics of apoptosis in HeLa cells were observed, using transmission electron microscopy. Loss of mitochondrial transmembrane potential was explored using laser scanning confocal microscopy with Rhodamine-123 (Rh123) staining. Furthermore, the mitochondrial apoptotic events were also confirmed by western blot analysis for the release of cytochrome C and apoptosis-inducing factor from mitochondria into the cytosol. In addition, activation of caspase-3, caspase-9, upregulation of Bax, p53 and downregulation of Bcl-2 were observed in HeLa cells also indicating apoptosis. Taken together, these results demonstrate that intense psPEF induce cell apoptosis through a mitochondrial-mediated pathway.

Optically monitoring voltage in neurons by photo-induced electron transfer through molecular wires

Fluorescence imaging is an attractive method for monitoring neuronal activity. A key challenge for optically monitoring voltage is development of sensors that can give large and fast responses to changes in transmembrane potential. We now present fluorescent sensors that detect voltage changes in neurons by modulation of photo-induced electron transfer (PeT) from an electron donor through a synthetic molecular wire to a fluorophore. These dyes give bigger responses to voltage than electrochromic dyes, yet have much faster kinetics and much less added capacitance than existing sensors based on hydrophobic anions or voltage-sensitive ion channels. These features enable single-trial detection of synaptic and action potentials in cultured hippocampal neurons and intact leech ganglia. Voltage-dependent PeT should be amenable to much further optimization, but the existing probes are already valuable indicators of neuronal activity.

Predicting Allosteric Pathways for Activation of K+ Channels by Perturbation Based Markovian Transmission Model

Kv1.2 voltage-gated and MlotiK1 cyclic nucleotide-gated K+ channels belong to the family of tetrameric cation channels and share a similar protein fold in the transmembrane region. Activation of both channels results in a passive flow of K+ ions, although Kv1.2 channel is activated by changes in transmembrane potential, while MlotiK1 channel is activated by the binding of cyclic nucleotides to the intracellular domain. Here we use a perturbation-based markovian transmission (PMT) model to study and compare allosteric activation pathways in Kv1.2 and MlotiK1 channels. With this model, the initial perturbation is converted to flow of probability, which allows studying of the time-course of signal transmission and propagation of probability flow through the protein molecule. We found that for the allosteric signal transmission to take place in Kv1.2, all ionizable residues in the TM region must be perturbed, while the perturbation of the ionizable residues on the S4 helix of Kv1.2 alone did not result in signal propagation. Furthermore, perturbation of ionizable residues in the TM region of cyclic nucleotide-gated MlotiK1 did not result in detectable signal transmission, while perturbation of the resolved part of the C-linker and the S2-S3 loop together initiate a signal transmission process, which went from the intracellular side of the channel to the extracellular side and to the filter and P-loop region. Our result showed that PMT model can distinguish different activation mechanisms in channels sharing similar fold. We have also predicted residues important for the allosteric signal transmission in Kv1.2 and MlotiK1 channels. Based on our calculations, we identified important structural elements in activation of MlotiK1 channel. In addition, we show that most of the on-pathway residues are aromatic residues in both channels.

Anticancer effect and mechanism of polymer micelle-encapsulated quercetin on ovarian cancer

Encapsulation of hydrophobic agents in polymer micelles can improve the water solubility of cargos, contributing to develop novel drugs. Quercetin (QU) is a hydrophobic agent with potential anticancer activity. In this work, we encapsulated QU into biodegradable monomethoxy poly(ethylene glycol)-poly(ε-caprolactone) (MPEG-PCL) micelles and tried to provide proof-of-principle for treating ovarian cancer with this nano-formulation of quercetin. These QU loaded MPEG-PCL (QU/MPEG-PCL) micelles with drug loading of 6.9% had a mean particle size of 36 nm, rendering the complete dispersion of quercetin in water. QU inhibited the growth of A2780S ovarian cancer cells on a dose dependent manner in vitro. Intravenous administration of QU/MPEG-PCL micelles significantly suppressed the growth of established xenograft A2780S ovarian tumors through causing cancer cell apoptosis and inhibiting angiogenesis in vivo. Furthermore, the anticancer activity of quercetin on ovarian cancer cells was studied in vitro. Quercetin treatment induced the apoptosis of A2780S cells associated with activating caspase-3 and caspase-9. MCL-1 downregulation, Bcl-2 downregulation, Bax upregulation and mitochondrial transmembrane potential change were observed, suggesting that quercetin may induce apoptosis of A2780S cells through the mitochondrial apoptotic pathway. Otherwise, quercetin treatment decreased phosphorylated p44/42 mitogen-activated protein kinase and phosphorylated Akt, contributing to inhibition of A2780S cell proliferation. Our data suggested that QU/MPEG-PCL micelles were a novel nano-formulation of quercetin with a potential clinical application in ovarian cancer therapy.

Experimental study on the photodynamic treatment of choroidal neovasculization with nanophthalocyanine photosensitizer.

To investigate the therapeutic effects of nanophthalocyanine photosensitizers on an experimental rat choroidal neovescularization (CNV) model, as well as to evaluate the cytotoxicity of which on human retinal pigment epithelia (HRPE) and human retinal endothelial cells (HRECs). Two types of photosensitizers, G(1)-ZnPc(COOH)(8) and G(1)-ZnPc(COOH)(8)/m respectively, were administrated for photodynamic therapy (PDT) after a successful establishment of CNV model on Brown-Norway (BN) rats via fundus photocoagulation. The therapeutic effects of the two drugs were assessed through optical coherence tomography (OCT), fluorescein fundus angiography (FFA) and transmission electron microscopy (TEM). For cytotoxicity tests, cell counting kit-8 (CCK-8) assays and changes of mitochondrial transmembrane potential (△Ψm) were conducted on HRPE and HRECs after initial uptake of the two drugs. Both photosensitizers demonstrated an improvement of vascular leakage and closure of CNV 1 week after PDT as confirmed by fundus image, OCT, FFA and TEM. Two weeks after PDT, G(1)-ZnPc(COOH)(8)/m showed a better CNV closure effect versus G(1)-ZnPc(COOH)(8) (P<0.05). A significant difference (P<0.01) was found in uptake of the two drugs in HRPE and HRECs, with no difference between the drugs (P>0.05). Both photosensitizers showed cytotoxicity on HRPE, but G(1)-ZnPc(COOH)(8)/m induced a lower cell viability. G(1)-ZnPc(COOH)(8)/m mediated PDT is better than G(1)-ZnPc(COOH)(8) in CNV closure and also have the advantage of fast metabolism leading to less side effect.

Transcranial static magnetic field stimulation in man: making things as simple as possible?

Transcranial static magnetic field stimulation in man: making things as simple as possible?

Transmembrane voltage potential controls embryonic eye patterning inXenopus laevis

Uncovering the molecular mechanisms of eye development is crucial for understanding the embryonic morphogenesis of complex structures, as well as for the establishment of novel biomedical approaches to address birth defects and injuries of the visual system. Here, we characterize change in transmembrane voltage potential (V(mem)) as a novel biophysical signal for eye induction in Xenopus laevis. During normal embryogenesis, a striking hyperpolarization demarcates a specific cluster of cells in the anterior neural field. Depolarizing the dorsal lineages in which these cells reside results in malformed eyes. Manipulating V(mem) of non-eye cells induces well-formed ectopic eyes that are morphologically and histologically similar to endogenous eyes. Remarkably, such ectopic eyes can be induced far outside the anterior neural field. A Ca(2+) channel-dependent pathway transduces the V(mem) signal and regulates patterning of eye field transcription factors. These data reveal a new, instructive role for membrane voltage during embryogenesis and demonstrate that V(mem) is a crucial upstream signal in eye development. Learning to control bioelectric initiators of organogenesis offers significant insight into birth defects that affect the eye and might have significant implications for regenerative approaches to ocular diseases.

Transcranial static magnetic field stimulation in man: Making things as simple as possible?

Transcranial static magnetic field stimulation in man: Making things as simple as possible?

Cytotoxicity of PP(Arg)(2)- and Hp(Arg)(2)-mediated photodynamic therapy and early stage of apoptosis induction in prostate carcinoma in vitro.

Porphyrin photosensitizers tend to localize in mitochondria. The depolarization of mitochondrial membrane is one of the early stages of apoptosis and Laser Scanning Fluorescence Microscopy allows to determine changes in transmembrane mitochondrial potential under influence of PDT depending on the kind of photosensitizer (PP(Arg)(2), Hp(Arg)(2)), the energy dose (5, 10, 30 and 50 J/cm(2)) and time periods (24 and 48 hours after irradiation) in the LNCaP (lymphonodal metastasis of prostate carcinoma, the androgen dependent cell line). Cyototoxicity induced by PP(Arg)(2)- and Hp(Arg)(2)-based PDT depending on energy dose and time after irradiation in prostate carcinoma is determined with MTT. Generally, it was shown that lower energy doses induce greater changes in transmembrane mitochondrial potential. Hp(Arg)(2)-based PDT was more effective causing greater mitochondrial membrane depolarization and cell viability decrease in comparison to PP(Arg)(2)-mediated PDT (in the case of maximal nontoxic photosensitizer doses used).

Small molecular anticancer agent SKLB703 induces apoptosis in human hepatocellular carcinoma cells via the mitochondrial apoptotic pathway invitro and inhibits tumor growth invivo

Inducing apoptosis is a promising therapeutic approach to overcome cancer. Here we described that a novel synthesized compound, 3-amino- N-(4-chlorobenzyl)-6-(3-methoxyphenyl)thieno[2,3- b]pyridine-2-carboxamide (SKLB703), exhibits antitumor activity via inducing apoptosis both in vitro and in vivo. Our results showed that SKLB703 inhibited the proliferation of a panel of human cancer cell lines, and human hepatocellular carcinoma cell line HepG2 was the most sensitive. The proliferation inhibitory effect of SKLB703 was associated with its apoptosis-inducing effect by activating caspase-3 and caspase-9 rather than caspase 8. Exposure of HepG2 to SKLB703 also resulted in Bax upregulation, Bcl-2 downregulation, cytochrome c release and mitochondrial transmembrane potential change in mitochondrial apoptotic pathway. Moreover, the decrease of phosphorylated p 44/42 mitogen-activated protein kinase and phosphorylated Akt was observed. SKLB703 suppressed the growth of established tumors in xenograft models in mice, whereas no toxicity was exhibited. TUNAL analysis showed that SKLB703 induced HepG2 tumor apoptosis. Taken together, the present study demonstrates that SKLB730 exhibits its antitumor activity through inducing apoptosis via mitochondrial apoptotic pathway. Its potential to be a candidate of anticancer agent is worth being further investigated.

Modelling the influence of thermal effects induced by radio frequency electric field on the dynamics of the ATPase nano-biomolecular motors

We model the dynamics of the F0 component of the F0F1-ATPase mitochondrion-based nano-motor operating in a stochastically-fluctuating medium that represents the intracellular environment. The stochastic dynamics are modeled via Langevin equation of motion wherein fluctuations are treated as white noise. We have investigated the influence of an applied alternating electric field on the rotary motion of the F0 rotor in such an environment. The exposure to the field induces a temperature rise in the mitochondrion’s membrane, within which the F0 is embedded. The external field also induces an electric potential that promotes a change in the mitochondrion’s transmembrane potential (TMP). Both the induced temperature and the change in TMP contribute to a change in the dynamics of the F0. We have found that for external fields in the radio frequency (RF) range, normally present in the environment and encountered by biological systems, the contribution of the induced thermal effects, relative to that of the induced TMP, to the dynamics of the F0 is more significant. The changes in the dynamics of the F0 part affect the frequency of the rotary motion of the F0F1-ATPase protein motor which, in turn, affects the production rate of the ATP molecules.

Guanylate Cyclase Activator YC-1 Enhances TRAIL-Induced Apoptosis in Human Epithelial Ovarian Carcinoma Cells via Activation of Apoptosis-Related Proteins

To assess the ability of 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) to promote apoptosis, we investigated the effect of YC-1 on tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in the human epithelial ovarian carcinoma cell lines. In OVCAR-3 and SK-OV-3 cell lines, we examined the stimulatory effect of YC-1 on TRAIL-induced apoptosis by monitoring cell death, nuclear damage, changes in apoptosis-related protein levels, activation of caspases and changes in the mitochondrial transmembrane potential. TRAIL induced a decrease in Bid, Bcl-2 and Bcl-xL protein levels, increase in cleaved Bid and Bax levels, loss of the mitochondrial transmembrane potential, cytochrome c release, activation of caspases (-8, -9 and -3) and an increase in the tumour suppressor p53 levels. YC-1 enhanced TRAIL-induced apoptosis-related protein activation, nuclear damage and cell death. Results from this study suggest that YC-1 may enhance the apoptotic effect of TRAIL on ovarian carcinoma cell lines by increasing the activation of the caspase-8- and Bid-dependent pathways and the mitochondria-mediated apoptotic pathway, leading to caspase activation. YC-1 may confer a benefit in TRAIL treatment of epithelial ovarian adenocarcinoma.

Plasma membrane charging of Jurkat cells by nanosecond pulsed electric fields

The initial effect of nanosecond pulsed electric fields (nsPEFs) on cells is a change of charge distributions along membranes. This first response is observed as a sudden shift in the plasma transmembrane potential that is faster than can be attributed to any physiological event. These immediate, yet transient, effects are only measurable if the diagnostic is faster than the exposure, i.e., on a nanosecond time scale. In this study, we monitored changes in the plasma transmembrane potential of Jurkat cells exposed to nsPEFs of 60ns and amplitudes from 5 to 90kV/cm with a temporal resolution of 5ns by means of the fast voltage-sensitive dye Annine-6. The measurements suggest the contribution of both dipole effects and asymmetric conduction currents across opposite sides of the cell to the charging. With the application of higher field strengths the membrane charges until a threshold voltage value of 1.4-1.6V is attained at the anodic pole. This indicates when the ion exchange rates exceed charging currents, thus providing strong evidence for pore formation. Prior to reaching this threshold, the time for the charging of the membrane by conductive currents is qualitatively in agreement with accepted models of membrane charging, which predict longer charging times for lower field strengths. The comparison of the data with previous studies suggests that the sub-physiological induced ionic imbalances may trigger other intracellular signaling events leading to dramatic outcomes, such as apoptosis.

Characterization of apoptotic changes induced by yessotoxin in the Bel7402 human hepatoma cell line

The apoptotic effects of yessotoxin (YTX) on Bel7402 human hepatoma cells have been evaluated by the combination of several methods, including optical microscopy, Hoechst 33342 staining and DNA gel electrophoresis. Rhodamine 123 staining has also been used to investigate changes in mitochondrial transmembrane potential. The results indicated that YTX has negative effects on human liver cells and induces apoptosis. Furthermore, changes in calcium concentrations were analyzed using the fluorescence probe Fluo-3 AM. The results showed that intracellular calcium concentrations increased after treatment with YTX.