Changes In Membrane Potential Research Articles

Reactive Oxygen Species Scavenging and Anti-Proliferative Potential of Veratric Acid: An in vitro Approach

Medicinal plants and their bioactive constituents play a vital role in the prevention of oxidative stress mediated diseases such as cancer and diabetes mellitus. The aim of this study is to investigate the reactive oxygen species (ROS) scavenging and anti-proliferative potential of veratric acid in vitro. The study analysed the antioxidant potential of veratric acid using in vitro free radical scavenging assays. The anti-proliferative potential of veratric acid was assessed by utilizing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, mitochondrial membrane potential (MMP) changes, intracellular ROS generation measurement and by determining the morphological alterations using Acridine orange/Ethidium bromide (Ao/EtBr) staining in the subline of Keratin-forming tumour cell line HeLa (KB). Veratric acid showed a good antioxidant activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), superoxide and hydroxyl radicals and the effect was found to be much comparable to that of the reference drug, ascorbic acid. Veratric acid significantly reduced the cell viability of KB cells and IC50 was 80 μg/ml. Veratric acid reduced the cell viability by generating excess ROS through activation of MMP depolarization and by inducing the apoptotic cell death of KB cells. The in vitro antioxidant and antiproliferative effect of veratric acid could be used further to validate its anti-carcinogenic potential using ideal experimental animal models.&nbsp

Bidirectional TRP/L Type Ca2+ Channel/RyR/BKCa Molecular and Functional Signaloplex in Vascular Smooth Muscles.

TRP channels are expressed both in vascular myocytes and endothelial cells, but knowledge of their operational mechanisms in vascular tissue is particularly limited. Here, we show for the first time the biphasic contractile reaction with relaxation followed by a contraction in response to TRPV4 agonist, GSK1016790A, in a rat pulmonary artery preconstricted with phenylephrine. Similar responses were observed both with and without endothelium, and these were abolished by the TRPV4 selective blocker, HC067047, confirming the specific role of TRPV4 in vascular myocytes. Using selective blockers of BKCa and L-type voltage-gated Ca2+ channels (CaL), we found that the relaxation phase was inducted by BKCa activation generating STOCs, while subsequent slowly developing TRPV4-mediated depolarisation activated CaL, producing the second contraction phase. These results are compared to TRPM8 activation using menthol in rat tail artery. Activation of both types of TRP channels produces highly similar changes in membrane potential, namely slow depolarisation with concurrent brief hyperpolarisations due to STOCs. We thus propose a general concept of bidirectional TRP-CaL-RyR-BKCa molecular and functional signaloplex in vascular smooth muscles. Accordingly, both TRPV4 and TRPM8 channels enhance local Ca2+ signals producing STOCs via TRP-RyR-BKCa coupling while simultaneously globally engaging BKCa and CaL channels by altering membrane potential.

An ultra-photostable and leakage-free fluorescent probe for long-term and high-fidelity mitochondria visualization and tracking

Mitochondria are important morphologically diverse organelles, and each characteristic has specific physiological significance. Thus, mitochondrial long-term and high-fidelity visualization and tracking are valuable. As a consequence, an ultra-photostable and leakage-free fluorescent probe is desired. Herein, we report a cationic probe DQPI-12 with a long alkyl chain, which can exclusively and firmly target mitochondria independent of mitochondria membrane potential changes. In particular, even after the cells have been fixed and left in place for 12 days, filamentous fluorescence signals from mitochondria could still be observed clearly, indicating DQPI-12 possesses excellent leakage-free performance. At the same time, compared with commercial mitochondria probes and those reported by others, DQPI-12 also displays excellent photostability. Inherent characters of DQPI-12 enable it to long-term and high-fidelity visualize mitochondria within a special microscopic field under continuous or intermittent irradiation of laser. Benefit from these advantages, DQPI-12 is successfully applied to track mitochondrial dynamic processes in living HeLa cells and neurons, and monitor the occurrence of mitophagy real-time, in-situ, and long-term.

The Mitochondrial Permeability Transition Pore-Current Knowledge of Its Structure, Function, and Regulation, and Optimized Methods for Evaluating Its Functional State.

The mitochondrial permeability transition pore (MPTP) is a calcium-dependent, ion non-selective membrane pore with a wide range of functions. Although the MPTP has been studied for more than 50 years, its molecular structure remains unclear. Short-term (reversible) opening of the MPTP protects cells from oxidative damage and enables the efflux of Ca2+ ions from the mitochondrial matrix and cell signaling. However, long-term (irreversible) opening induces processes leading to cell death. Ca2+ ions, reactive oxygen species, and changes in mitochondrial membrane potential regulate pore opening. The sensitivity of the pore to Ca2+ ions changes as an organism ages, and MPTP opening plays a key role in the pathogenesis of many diseases. Most studies of the MPTP have focused on elucidating its molecular structure. However, understanding the mechanisms that will inhibit the MPTP may improve the treatment of diseases associated with its opening. To evaluate the functional state of the MPTP and its inhibitors, it is therefore necessary to use appropriate methods that provide reproducible results across laboratories. This review summarizes our current knowledge of the function and regulation of the MPTP. The latter part of the review introduces two optimized methods for evaluating the functional state of the pore under standardized conditions.

The interplay of intracellular calcium and zinc ions in response to electric field stimulation in primary rat cortical neurons in vitro.

Recent pharmacological studies demonstrate a role for zinc (Zn2+) in shaping intracellular calcium (Ca2+) dynamics and vice versa in excitable cells including neurons and cardiomyocytes. Herein, we sought to examine the dynamic of intracellular release of Ca2+ and Zn2+ upon modifying excitability of primary rat cortical neurons using electric field stimulation (EFS) in vitro. We show that exposure to EFS with an intensity of 7.69 V/cm induces transient membrane hyperpolarization together with transient elevations in the cytosolic levels of Ca2+ and Zn2+ ions. The EFS-induced hyperpolarization was inhibited by prior treatment of cells with the K+ channel opener diazoxide. Chemical hyperpolarization had no apparent effect on either Ca2+ or Zn2+. The source of EFS-induced rise in Ca2+ and Zn2+ seemed to be intracellular, and that the dynamic inferred of an interplay between Ca2+ and Zn2+ ions, whereby the removal of extracellular Ca2+ augmented the release of intracellular Ca2+ and Zn2+ and caused a stronger and more sustained hyperpolarization. We demonstrate that Zn2+ is released from intracellular vesicles located in the soma, with major co-localizations in the lysosomes and endoplasmic reticulum. These studies further support the use of EFS as a tool to interrogate the kinetics of intracellular ions in response to changing membrane potential in vitro.

Effects of different doses of dopamine receptor agonist pramipexole on neurobehaviors and changes of mitochondrial membrane potentials in rats with global cerebral ischemia-reperfusion injury

ObjectiveTo explore the effects of different doses of dopamine receptor agonist pramipexole on neurobehaviors and changes of mitochondrial membrane potential in rats with global cerebral ischemia-reperfusion injury. MethodsA total of 75 SPF Sprague-Dawley male rats were randomly divided into sham group (n=20), model group (n=20), pramipexole administration group (n=35). The rat model of global cerebral ischemia-reperfusion injury was prepared by the modified Pulsinelli's four-vessel occlusion method. Pramipexole administration group was administered intraperitoneally in rats with global cerebral ischemia-reperfusion injury at different doses of pramipexole 0.25 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, once a day for 14 consecutive days. Based on the results of modified neurological severity scores, open field test and morphology by Nissl's staining to determine the optimal dose of pramipexole. Mitochondrial membrane potential in the optimal dose of pramipexole administration group were measured by the JC-1 fluorescent probe staining method. Results1. Different doses of pramipexole 0.25 mg/kg, 0.5 mg/kg, 1 mg/kg, and 2 mg/kg, were used as drug administration in rats with global cerebral ischemia-reperfusion injury for 14 consecutive days, and we found that all four doses of pramipexole could improve the modified neurological severity scores of rats with global cerebral ischemia-reperfusion injury to varying degrees, but only 0.5 mg/kg pramipexole at 1, 3, 7 and 14 days consistently reduced modified neurological severity scores and improved neurological function in rats with global cerebral ischemia-reperfusion injury. In the open-field test, only 0.5 mg/kg pramipexole increased the number of entries into the central zone, duration spent in the central zone, total distance travelled in the open field and average velocity, which improved the spontaneous activities and reduced anxiety and depression of rats with global cerebral ischemia-reperfusion injury. 2. Different doses of pramipexole 0.25 mg/kg, 0.5 mg/kg, 1 mg/kg, and 2 mg/kg for 14 consecutive days significantly increased the number of surviving neurons in the hippocampal CA1 subfield in rats with global cerebral ischemia-reperfusion injury to varying degrees. Based on these results, we tentatively found that 0.5 mg/kg pramipexole may be the optimal dose in all of the above. 3. We found that 0.5 mg/kg pramipexole significantly increased the mitochondrial membrane potential in rats after global cerebral ischemia-reperfusion injury. ConclusionDifferent doses of dopamine receptor agonist pramipexole improved neurological function of rats with global cerebral ischemia-reperfusion injury to varying degrees, and 0.5 mg/kg pramipexole may be the optimal dose in all of the above. Pramipexole may produce neuroprotective effects by protecting neurons in the hippocampus and improving the mitochondrial membrane potential after global cerebral ischemia-reperfusion injury.

Synthesis, characterization, anticancer efficacy evaluation of ruthenium(II) and iridium(III) polypyridyl complexes toward A549 cells.

A new ligand DFIP (2-(dibenzo[b,d]furan-3-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) and its two complexes iridium(III) [Ir(ppy)2(DFIP)](PF6) (ppy = 2-phenylpyridine, Ir1) and ruthenium(II) [Ru(bpy)2(DFIP)](PF6)2 (bpy = 2,2'-bipyridine, Ru1) were synthesized and characterized. The anticancer effects of the two complexes on A549, BEL-7402, HepG2, SGC-7901, HCT116 and normal LO2 cells were tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Complex Ir1 shows high cytotoxic activity on A549, BEL-7402, SGC-7901 and HepG2, Ru1 exhibits moderate anticancer activity toward A549, BEL-7402 and SGC-7901 cells. The IC50 values of Ir1 and Ru1 toward A549 are 7.2 ± 0.1 and 22.6 ± 1.4μM, respectively. The localization of complexes Ir1 and Ru1 in the mitochondrial, intracellular accumulation of reactive oxygen species (ROS) levels, and the changes of mitochondrial membrane potential (MMP) and cytochrome c (cyto-c) were investigated. Apoptosis and cell cycle were detected by flow cytometry. Immunogenic cell death (ICD) was used to detect the effects of Ir1 and Ru1 on the A549 using a confocal laser scanning microscope. The expression of apoptosis-related proteins was detected by western blotting. Ir1 and Ru1 can increase the intracellular ROS levels and release cyto-c, reduce the MMP, leading to the apoptosis of A549 cells and blocking the A549 cells at the G0/G1 phase. Additionally, the complexes caused a decrease of the expression of polyADP-ribose polymerase (PARP), caspase 3, Bcl-2 (B-cell lymphoma-2), PI3K (phosphoinositide-3 kinase) and upregulated the expression of Bax. All these findings indicated that the complexes exert anticancer efficacy to induce cell death through immunogenic cell death, apoptosis, and autophagy.

Novel Property Cytotoxicity and Mechanism of Food Preservative Brevilaterins against Human Gastric Cancer Cells.

Brevilaterins, antimicrobial peptides produced by Brevibacillus laterosporus, are regarded as excellent food preservatives and are popular as antimicrobial applications. Recent research has uncovered their potent cytotoxic effects against diverse cancer cells, thereby underscoring the pressing need for more extensive and intensive investigations into this use. In this study, we explored their novel function in inducing cytotoxicity to cancer cells and systematically investigated the mechanism of action of Brevilaterin B/C (BB/BC) in vivo. Proliferation, membrane permeability, and apoptotic rate were evaluated using CCK-8 assay, LDH assay, and Annexin V-FITC/PI kits. ROS levels and mitochondrial membrane potential were detected using the fluorescent probe DCFH-DA and JC-1. Our results demonstrated that both BB and BC at concentrations of 4-6 µg/mL significantly inhibited the proliferation and migration of gastric cancer cells BGC-823. Treatment with 4 µg/mL of BB/BC rapidly increased LDH levels in the supernatant of BGC-823 cells, leading to further investigation of the mechanism of apoptosis. We found that the apoptotic rate of BGC-823 cells significantly increased upon treatment with BB/BC, demonstrating their potent induction of apoptosis. BB/BC-induced ROS production in BGC-823 cells impaired their growth and induced apoptosis, indicating a close association between apoptosis and ROS elevation. Additionally, JC-1 aggregates rapidly accumulated after treatment with 4 µg/mL of BB/BC, suggesting changes in mitochondrial membrane potential and early apoptosis. Taken together, our findings revealed that BB and BC exhibit significant anticancer effects against gastric cancer cells, highlighting the promising potential of Brevilaterins as anticancer agents.

The mechanism by which miR-494-3p regulates PGC1-α-mediated inhibition of mitophagy in cardiomyocytes and alleviation of myocardial ischemia—reperfusion injury

ObjectiveThe purpose of this study was to explore whether miR-494-3p inhibits the occurrence of mitochondrial autophagy in cardiomyocytes by inhibiting the expression of PGC1-α and to supplement the theoretical basis for the role of autophagy in cardiac injury induced by hypoxia/reperfusion (H/R).MethodsThe expression of miR-494-3p was detected by RT‒qPCR, and the expression of PGC1-α, autophagy-related proteins (LC3, Beclin 1), apoptosis-related proteins (Bax and Bcl-2), PINK1/Parkin signaling pathway-related proteins (PINK1, Parkin) and mitochondrial change-related proteins (Mfn1, Mfn2, OPA1) was detected by Western blotting. The changes in mitochondrial membrane potential were detected by JC-1 staining (ΔΨm). The formation of autophagosomes was observed by transmission electron microscopy. Cell proliferation activity was detected by CCK-8, and cell apoptosis was detected by flow cytometry. A dual-luciferase gene reporter assay identified a targeted binding site between miR-494-3p and PGC1-α.ResultsThe results showed that miR-494-3p and PGC1-α were differentially expressed in H/R cardiomyocytes; that is, the expression of miR-494-3p was downregulated, and the expression of PGC1-α was upregulated. In addition, mitochondrial autophagy occurred in H/R cardiomyocytes. That is, LC3-II/LC3-I, Beclin 1, PINK1, and Parkin expression was upregulated, Mfn1, Mfn2, and OPA1 expression was downregulated, and the mitochondrial membrane potential was decreased. The transfection of miR-494-3p mimic can significantly improve the cell proliferation activity of cardiomyocytes and inhibit the occurrence of cardiomyocyte apoptosis and autophagy, while the transfection of miR-494-3p inhibitor has the opposite result. After transfection of the miR-494-3p mimic, treatment with autophagy inhibitors and activators changed the effects of miR-494-3p on cardiomyocyte proliferation and apoptosis. At the same time, the overexpression of PGC1-α reversed the promoting effect of miR-494-3p on cardiomyocyte proliferation and the inhibitory effect on apoptosis and autophagy.ConclusionMiR-494-3p can target and negatively regulate the expression of PGC1-α to inhibit mitophagy in cardiomyocytes.

The bioenergetic "CK Clamp" technique detects substrate-specific changes in mitochondrial respiration and membrane potential during early VML injury pathology.

Volumetric muscle loss (VML) injuries are characterized by non-recoverable loss of tissue resulting in contractile and metabolic dysfunction. The characterization of metabolic dysfunction in volumetric muscle loss-injured muscle has been interpreted from permeabilized myofiber respiration experiments involving saturating ADP levels and non-physiologic ATP:ADP concentration ratios. The extent to which this testing condition obscures the analysis of mitochondrial (dys) function after volumetric muscle loss injury is unclear. An alternative approach is described that leverages the enzymatic reaction of creatine kinase and phosphocreatine to assess mitochondrial respiration and membrane potential at clamped physiologic ATP:ADP ratios, "CK Clamp." The objective of this study was to validate the CK Clamp in volumetric muscle loss-injured muscle and to detect differences that may exist between volumetric muscle loss-injured and uninjured muscles at 1, 3, 5, 7, 10, and 14days post-injury. Volumetric muscle loss-injured muscle maintains bioenergetic features of the CK Clamp approach, i.e., mitochondrial respiration rate (JO2) titters down and mitochondrial membrane potential is more polarized with increasing ATP:ADP ratios. Pyruvate/malate/succinate-supported JO2 was significantly less in volumetric muscle loss-injured muscle at all timepoints compared to uninjured controls (-26% to -84%, p < 0.001) and electron conductance was less at day 1 (-60%), 5 (-52%), 7 (-35%), 10 (-59%), and 14 (-41%) (p < 0.001). Palmitoyl-carnitine/malate-supported JO2 and electron conductance were less affected following volumetric muscle loss injury. volumetric muscle loss-injury also corresponded with a more polarized mitochondrial membrane potential across the clamped ATP:ADP ratios at day 1 and 10 (pyruvate and palmitoyl-carnitine, respectively) (+5%, p < 0.001). This study supports previous characterizations of metabolic dysfunction and validates the CK Clamp as a tool to investigate bioenergetics in traumatically-injured muscle.

Evaluation of the Anticancer Activity and Mechanism Studies of Glycyrrhetic Acid Derivatives toward HeLa Cells.

In this paper, a series of glycyrrhetic acid derivatives 3a-3f were synthesized via the esterification reaction. The cytotoxicity of these compounds against five tumor cells (SGC-7901, BEL-7402, A549, HeLa and B16) and normal LO2 cells was investigated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The results showed that compound 3a exhibited high antiproliferative activity against HeLa cells (IC50 = 11.4 ± 0.2 μM). The anticancer activity was studied through apoptosis, cloning, and scratching; the levels of the intracellular ROS, GSH, and Ca2+; and the change in the mitochondrial membrane potential, cell cycle arrest and RNA sequencing. Furthermore, the effects of compound 3a on gene expression levels and metabolic pathways in HeLa cells were investigated via transcriptomics. The experimental results showed that this compound can block the cell cycle in the S phase and inhibit cell migration by downregulating Focal adhesion kinase (FAK) expression. Moreover, the compound can reduce the intracellular glutathione (GSH) content, increase the Ca2+ level and the intracellular ROS content, and induce a decrease in the mitochondrial membrane potential, further leading to cell death. In addition, it was also found that the mechanism of compounds inducing apoptosis was related to the regulation of the expression of mitochondria-related proteins B-cell lymphoma-2 (Bcl-2), Bcl-2-Associated X (Bax), and the activation of the caspase proteins. Taken together, this work provides a help for the development of glycyrrhetinic acid compounds as potential anticancer molecules.

Berberine disrupts staphylococcal proton motive force to cause potent anti-staphylococcal effects in vitro

The presence of antibiotic resistance has increased the urgency for more effective treatments of bacterial infections. Biofilm formation has complicated this issue as biofilm bacteria become tolerant to antibiotics due to environmental factors such as nutrient deprivation and adhesion. In septic arthritis, a disease with an 11% mortality rate, bacteria in synovial fluid organize into floating, protein-rich, bacterial aggregates (mm-cm) that display depressed metabolism and antibiotic tolerance. In this study, Staphylococcus aureus (S. aureus), which is the most common pathogen in septic arthritis, was tested against different inhibitors that modulate bacterial surface protein availability and that should decrease bacterial aggregation. One of these, berberine, a quaternary ammonium compound, was found to reduce bacterial counts by 3–7 logs in human synovial fluid (aggregating medium) with no effect in tryptic soy broth (TSB, non-aggregating). Unlike traditional antibiotics, the bactericidal activity of berberine appeared to be independent of bacterial metabolism. To elucidate the mechanism, we used synovial fluid fractionation, targeted MRSA transposon insertion mutants, dyes to assess changes in membrane potential (DiSC3(5)) and membrane permeability (propidium iodide (PI)), colony counting, and fluorescence spectroscopy. We showed that berberine's activity was dependent on an alkaline pH and berberine killed both methicillin-sensitive S. aureus and MRSA in alkaline media (pH 8.5–9.0; p < 0.0001 vs. same pH controls). Under these alkaline conditions, berberine localized to S. aureus where berberine was isolated in cytoplasmic (∼95%) and DNA (∼5%) fractions. Importantly, berberine increased bacterial cell membrane permeability, and disrupted the proton motive force, suggesting a mechanism whereby it may be able to synergize with other antibacterial compounds under less harsh conditions. We suggest that berberine, which is cheap and readily available, can be made into an effective treatment.

Salidroside Attenuates Cisplatin-Induced Ototoxicity: An Experimental Study In Vitro and In Vivo

Oxidative damage to hair cells is the major cause of ototoxicity induced by cisplatin (cis)-based chemotherapy. In this study, we aimed to assess how salidroside (SAL) protected cochlear explants (CEs) and HEI-OC1 cell lines against cis-induced ototoxicity and reduced relative hearing loss in mouse models. Furthermore, the protective mechanism of the Nrf2/ARE pathway was investigated. Cell Counting Kit-8 was used to measure the viability of HEI-OC1 cells. Flow cytometry and the TUNEL assay were used to evaluate cell apoptosis. Flow cytometry was used to measure intracellular reactive oxygen species (ROS). Immunofluorescence staining determined the changes in mitochondrial membrane potential (ΔΨm). Western blot was used to measure the levels of caspase-3 and Nrf-2. An analysis of Nrf2 and target gene levels of expression was conducted using qRT-PCR. Hearing was monitored using auditory brainstem response audiometry. In cochlear explants, SAL inhibits cis-induced apoptosis of HEI-OC1 cells and decreased hair cell apoptosis. SAL inhibited cis-induced apoptosis by lowering intracellular ROS, preserving mitochondrial function, and reducing caspase-3 expression. Moreover, auditory cells were protected from the toxic effects of cis by the Nrf2-ARE pathway after treatment with SAL. In Vivo, SAL could protect against cis-induced hearing loss, and the use of the PLGA-poloxamer nanohydrogel as a carrier increased the protection efficiency of SAL. Through its ability to reduce oxidative stress, SAL could protect auditory cell lines from cis-induced apoptosis In Vitro and attenuate cis-induced hearing loss In Vivo. Nano-based drug delivery can improve the protection efficiency of SAL. Further research should be conducted on the antioxidant capacity of SAL and its use in ototoxicity.

Ion Channels as a Potential Target in Pharmaceutical Designs

Voltage-gated ion channels are integral membrane proteins that respond to changes in membrane potential with rapid variations in membrane permeability to ions [...].

Synthesis and characterization of novel Schiff base-silicon (IV) phthalocyanine complex for photodynamic therapy of breast cancer cell lines

BackgroundPhotodynamic therapy is an alternative anticancer treatment approach that promises high therapeutic efficacy. In this study, it is aimed to investigate the PDT-mediated anticancer effects of newly synthesized silicon phthalocyanine (SiPc) molecules on MDA-MB-231, MCF-7 breast cancer cell lines, and non-tumorigenic MCF-10A breast cell line. MethodsNovel bromo substituted Schiff base (3a), its nitro homolog (3b), and their silicon complexes (SiPc-5a and SiPc-5b) were synthesized. Their proposed structures were confirmed by FT-IR, NMR, UV–vis and MS instrumental techniques. MDA-MB-231, MCF-7 and MCF-10A cells were illuminated at a light wavelength of 680 nm for 10 min, giving a total irradiation dose of 10 j/cm2. MTT assay was used to determine the cytotoxic effects of SiPc-5a and SiPc-5b. Apoptotic cell death was analyzed using flow cytometry. Changes in the mitochondrial membrane potential were determined by TMRE staining. Intracellular ROS generation was observed microscopically using H2DCFDA dye. Colony formation assay and in vitro scratch assay were performed to analyze the clonogenic activity and cell motility. Transwell migration and matrigel invasion analyzes were conducted to observe changes in the migration and invasion status of the cells. ResultsThe combination of SiPc-5a and SiPc-5b with PDT exhibited cytotoxic effects on cancer cells and triggered cell death. SiPc-5a/PDT and SiPc-5b/PDT decreased mitochondrial membrane potential and increased intracellular ROS production. Statistically significant changes were detected in cancer cells' colony-forming ability and motility. SiPc-5a/PDT and SiPc-5b/PDT reduced cancer cells' migration and invasion capacities. ConclusionThe present study identifies PDT-mediated antiproliferative, apoptotic, and anti-migratory characteristics of novel SiPc molecules. The outcomes of this study emphasize the anticancer properties of these molecules and suggest that they may be evaluated as drug-candidate molecules for therapeutic purposes.

The SLC9C2 Gene Product (Na+/H+ Exchanger Isoform 11; NHE11) Is a Testis-Specific Protein Localized to the Head of Mature Mammalian Sperm.

Na+/H+ exchangers (NHEs) are a family of ion transporters that regulate the pH of various cell compartments across an array of cell types. In eukaryotes, NHEs are encoded by the SLC9 gene family comprising 13 genes. SLC9C2, which encodes the NHE11 protein, is the only one of the SLC9 genes that is essentially uncharacterized. Here, we show that SLC9C2 exhibits testis/sperm-restricted expression in rats and humans, akin to its paralog SLC9C1 (NHE10). Similar to NHE10, NHE11 is predicted to contain an NHE domain, a voltage sensing domain, and finally an intracellular cyclic nucleotide binding domain. An immunofluorescence analysis of testis sections reveals that NHE11 localizes with developing acrosomal granules in spermiogenic cells in both rat and human testes. Most interestingly, NHE11 localizes to the sperm head, likely the plasma membrane overlaying the acrosome, in mature sperm from rats and humans. Therefore, NHE11 is the only known NHE to localize to the acrosomal region of the head in mature sperm cells. The physiological role of NHE11 has yet to be demonstrated but its predicted functional domains and unique localization suggests that it could modulate intracellular pH of the sperm head in response to changes in membrane potential and cyclic nucleotide concentrations that are a result of sperm capacitation events. If NHE11 is shown to be important for male fertility, it will be an attractive target for male contraceptive drugs due to its exclusive testis/sperm-specific expression.

The surface conductance of red blood cells and platelets is modulated by the cell membrane potential.

Dielectrophoretic analysis of cell electrical properties via the Clausius-Mossotti model has been widely used to estimate values of the membrane conductance, membrane capacitance and cytoplasm conductivity of cells. However, although the latter two values produced by this method compare well to those acquired by other electrophysiological methods, the membrane conductance is often substantially larger than that acquired by methods such as patch clamp. In this paper, the electrical properties of red blood cells (RBC) are analysed at two conductivities and following membrane-altering treatments, to develop a mathematical model of membrane conductance. Results suggest that the RBC "membrane conductance" term is primarily dominated by surface conduction, comprising an element related to medium conductivity augmented by conduction in the electrical double layer, which is in turn altered by the cell membrane potential. Validation of the relationship between membrane potential and membrane conductance was performed using platelets, where a similar relationship was observed. This sheds new light on the origin and significance of the membrane conductance term and explains for the first time phenomena of alterations in the parameter counter to changes in membrane potential or cytoplasm conductivity.

Ribbon Synapses and Retinal Disease: Review.

Synaptic ribbons are presynaptic protein complexes that are believed to be important for the transmission of sensory information in the visual system. Ribbons are selectively associated with those synapses where graded changes in membrane potential drive continuous neurotransmitter release. Defective synaptic transmission can arise as a result of the mutagenesis of a single ribbon component. Visual diseases that stem from malfunctions in the presynaptic molecular machinery of ribbon synapses in the retina are rare. In this review, we provide an overview of synaptopathies that give rise to retinal malfunction and our present understanding of the mechanisms that underlie their pathogenesis and discuss muscular dystrophies that exhibit ribbon synapse involvement in the pathology.

Comparative efficacy of epigallocatechin gallate and its nano-formulation in prostate cancer 3D spheroids model

BackgroundProstate cancer (PCa) remains one of the clinically relevant pathologies that needs pragmatic and effective treatment approaches. The current study aimed to evaluate the anticancer potential of epigallocatechin gallate (EGCG) and EGCG-loaded nanoparticles (EGCG NP) for the treatment of prostate cancer in in-vitro 3D spheroid model. MethodsThe EGCG NPs was synthesized by using polymeric method as reported in our previous study. A 3D spheroid assay was conducted using human prostate specific cell lines (PC3 and 22Rv1) cultured on poly-HEMA-covered plates at different time points. Once formed, the spheroids were treated with either EGCG alone or with EGCG NPs continuously for 6 days. Simultaneously, specific controls were also taken for comparison purpose. CellROX dye was used to quantitate the formation of reactive oxygen species (ROS) in response to EGCG and EGCG NPs to 22Rv1 and PC3, 3D spheroids. The treated spheroids were also evaluated to measure modulation in mitochondrial membrane potential and the quantification of apoptotic and live cells using a flow cytometer. ResultsThe spheroid sizes of both studied cell lines were found to be significantly (p < 0.05) reduced after the treatment with EGCG and its nanoformulation. The significant increase in ROS formation was observed in PC3 cells in response to EGCG and EGCG-NPs treatment. However, no significant change in ROS formation was observed in 22Rv1 cells. Similarly, both compounds (EGCG and EGCG NPs) did not show any significant changes in mitochondrial membrane potential in 22Rv1 and PC3 spheroids. Interestingly, EGCG treatment showed a significant change between live and apoptotic cells in both 22Rv1 and PC3 spheroids but its nanoformulation didn’t show any significant change in the number of apoptotic and live cells. Our study observed a significant anticancer potential of EGCG and EGCG NPs at clinically relevant doses highlighting the possible advantage of 3D spheroid model specifically in our studied cancer cells. However, further preclinical in vivo studies are recommended in a suitable model to decipher our in vitro data and exploit EGCG and EGCG NPs against prostate cancer. ConclusionOur results indicate the anticancer efficacy of EGCG and EGCG NPs in 3D spheroids of PCa cell lines.

Study on the effect of 5-aminolevulinic acid-mediated photodynamic therapy combined with cisplatin on human ovarian cancer OVCAR-3 ​cells

PurposeThis article explores the effect of 5-aminolevulinic acid (5-ALA)-mediated photodynamic therapy (PDT) combined with cisplatin (CDDP) on the apoptosis of human ovarian cancer cells and the mechanism of action of the combination therapy. Materials and methodsHuman ovarian cancer OVCAR-3 ​cells were cultured in vitro and divided into 5-ALA/PDT group, CDDP group and combined treatment group (5-ALA/PDT combined with different concentrations of CDDP). After administration of the corresponding drugs, a CCK-8 assay was used to detect the inhibition rate of cell proliferation. After Rhodamine 123 staining, mitochondrial membrane potential changes were observed under fluorescence microscopy. The apoptosis rate and reactive oxygen species (ROS) content were detected by flow cytometry. Western blotting was used to detect protein expression. ResultsThe CCK-8 assay showed that CDDP in combination with 5-ALA/PDT significantly enhanced cytotoxicity compared to treatment with CDDP alone and that low doses of CDDP were sufficient to induce these combination effects. The mitochondrial membrane potential in each combination treatment group gradually decreased with increasing CDDP concentration, while the apoptosis rate and reactive oxygen species (ROS) content detected by flow cytometry gradually increased. Western blotting assay showed that the expression of bax, cleaved caspase-9, cleaved caspase-3, and cleaved PARP was increased, while the expression of bcl-2, caspase-9, caspase-3, and PARP was decreased, and the differences were statistically significant (P ​< ​0.05). ConclusionsIn summary, 5-ALA/PDT combined with CDDP can effectively inhibit cell proliferation and promote apoptosis, and this combination may induce apoptosis by activating the mitochondrial pathway.