Increased Propidium Iodide Uptake Research Articles

Abstract 4333: Identification of glucose-6-phosphate dehydrogenase dependency in IDH1 mutant glioma cells using functional genomics

Abstract Astrocytoma and oligodendroglioma are most commonly initiated by missense mutations at the arginine 132 codon of isocitrate dehydrogenase 1 (IDH1). Biochemically the mutant IDH1 protein acquires neomorphic activity: reductive NADPH-dependent catalysis of α-ketoglutarate (αKG) to (R)-2-hydroxyglutarate [(R)-2-HG]. (R)-2-HG interferes with the function of αKG-dependent epigenetic modifiers, resulting in extensive epigenetic remodeling that impairs cellular differentiation and promotes gliomagenesis. Strategies for selectively targeting IDH1-mutant cells offer promise for improved treatment over the standard modalities of surgery, chemotherapy and radiotherapy. Whole-genome CRISPR/Cas9 knockout screens were conducted to identify gene dependencies in isogenic IDH1-mutant and wild-type (WT) U-87 MG cells. Cells were transduced with the Brunello single guide RNA (sgRNA) library, grown for 21 days, then sgRNA distributions were assessed by amplicon sequencing. Gene dependencies were identified using CRISPRcleanR and BAGELR pipelines. We discovered that glucose-6-phosphate dehydrogenase (G6PD), a pentose phosphate pathway (PPP) enzyme, was needed for viability of IDH1-mutant cells, but was dispensable for U-87 MG WT cells. The PPP is the main producer of cytosolic NADPH that is required for de novo lipogenesis, maintenance of antioxidants and production of (R)-2-HG by mutant IDH1. Previous reports demonstrated that IDH1-mutant cells increase PPP flux to support these processes. We have validated this dependency using G6PDi-1, a cell-active inhibitor of G6PD. Growth inhibition (IC50) assays demonstrated that IDH1-mutant U-87 MG cells were threefold more sensitive to G6PDi-1 with IC50 of 23 μM and enhanced cell death (increased propidium iodide uptake), compared with WT U-87 MG cells with IC50 of 67 μM. G6PD knockout was then conducted to further validate this dependency. Nucleofection of IDH1 WT U-87 MG cells with G6PD multi guide RNA-Cas9 ribonucleoproteins resulted in highly efficient knockout of G6PD at day 5 (99-100%), which was maintained at day 21 (96-98%). In contrast, efficient G6PD knockout in IDH1-mutant U-87 MG cells at day 5 (98-100%) was lost following 21 days culture (0-5%), indicating death of almost all edited cells within the culture. Finally, Kaplan-Meier analysis of IDH1-mutant patients in the TCGA lower grade glioma dataset demonstrated that higher G6PD expression correlated with worse median overall survival (7.3 versus 9.5 years; log-rank p=0.01), but was not prognostic in the IDH1 WT TCGA glioblastoma multiforme dataset. These findings demonstrate that a cell line model of IDH1-mutant glioma is highly dependent on G6PD for survival. This observation builds on the recognized importance of the PPP in the metabolic biology of IDH1-mutant malignancy by establishing G6PD as a targetable dependency in this disease. Citation Format: Kim M. Paras, Pooja Patel, Paul Choi, William R. Wilson, Tet-Woo Lee, Stephen M. Jamieson, Dean C. Singleton. Identification of glucose-6-phosphate dehydrogenase dependency in IDH1 mutant glioma cells using functional genomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4333.

"Antibacterial effect and possible mechanism of action of 1,3,4-oxadiazole in Staphylococcus aureus".

Staphylococcus aureus is one of the main etiological agents causing foodborne diseases, and the development of new antibacterial agents is urgent. This study evaluated the antibacterial activity and the possible mechanism of action of the 1,3,4-oxadiazole LMM6 against S. aureus. The minimum inhibitory concentration (MIC) of LMM6 ranged from 1.95 to 7.81µg ml-1. The time-kill assay showed that 48-h treatment at 1×to 8×MIC reduced S. aureus by 4 log colony forming unit (CFU), indicating a bacteriostatic effect. Regarding the possible mechanism of action of LMM6, there was accumulation of reactive oxygen species (ROS) and an increase in the absorption of crystal violet (∼50%) by the cells treated with LMM6 at 1×and 2×MIC for 6-12h. In addition, there was increased propidium iodide uptake (∼84%) after exposure to LMM6 for 12h at 2×MIC. After 48h of treatment, 100% of bacteria had been injured. Scanning electron microscopy observations demonstrated that LMM6-treated cells were smaller compared with the untreated group. LMM6 exhibited bacteriostatic activity and its mechanism of action involves increase of intracellular ROS and disturbance of the cell membrane, which can be considered a key target for controlling the growth of S. aureus.

GJB4 variants linked to skin disease exhibit a trafficking deficiency en route to gap junction formation that can be restored by co-expression of select connexins.

Epidermal keratinocytes are enriched with at least nine connexins that are key regulators of epidermal homeostasis. The role of Cx30.3 in keratinocytes and epidermal health became evident when fourteen autosomal dominant mutations in the Cx30.3-encoding GJB4 gene were linked to a rare and incurable skin disorder called erythrokeratodermia variabilis et progressiva (EKVP). While these variants are linked to EKVP, they remain largely uncharacterized hindering therapeutic options. In this study, we characterize the expression and functional status of three EKVP-linked Cx30.3 mutants (G12D, T85P, and F189Y) in tissue-relevant and differentiation-competent rat epidermal keratinocytes. We found that GFP-tagged Cx30.3 mutants were non-functional likely due to their impaired trafficking and primary entrapment within the endoplasmic reticulum (ER). However, all mutants failed to increase BiP/GRP78 levels suggesting they were not inducing an unfolded protein response. FLAG-tagged Cx30.3 mutants were also trafficking impaired yet occasionally exhibited some capacity to assemble into gap junctions. The pathological impact of these mutants may extend beyond their trafficking deficiencies as keratinocytes expressing FLAG-tagged Cx30.3 mutants exhibited increased propidium iodide uptake in the absence of divalent cations. Attempts to rescue the delivery of trafficking impaired GFP-tagged Cx30.3 mutants into gap junctions by chemical chaperone treatment were ineffective. However, co-expression of wild type Cx30.3 greatly enhanced the assembly of Cx30.3 mutants into gap junctions, although endogenous levels of Cx30.3 do not appear to prevent the skin pathology found in patients harboring these autosomal dominant mutations. In addition, a spectrum of connexin isoforms (Cx26, Cx30, and Cx43) exhibited the differential ability to trans-dominantly rescue the assembly of GFP-tagged Cx30.3 mutants into gap junctions suggesting a broad range of connexins found in keratinocytes may favourably interact with Cx30.3 mutants. We conclude that selective upregulation of compatible wild type connexins in keratinocytes may have potential therapeutic value in rescuing epidermal defects invoked by Cx30.3 EKVP-linked mutants.

The elucidation of the multimodal action of the investigational anti-Candida lipopeptide (AF4) lead from Bacillus subtilis.

Background: Candida species are the main etiological agents for candidiasis, and Candida albicans are the most common infectious species. Candida species' growing resistance to conventional therapies necessitates more research into novel antifungal agents. Antifungal peptides isolated from microorganisms have potential applications as novel therapeutics. AF4 a Bacillus-derived lipopeptide demonstrating broad-spectrum antifungal activity has been investigated for its ability to cause cell death in Candida species via membrane damage and oxidative stress. Methods: Using biophysical techniques, the secondary structure of the AF4 lipopeptide was identified. Scanning electron microscopy and confocal microscopy with fluorescent dyes were performed to visualise the effect of the lipopeptide. The membrane disruption and permeabilization were assessed using the 1,6-diphenyl hexatriene (DPH) fluorescence assay and flow cytometric (FC) assessment of propidium iodide (PI) uptake, respectively. The reactive oxygen species levels were estimated using the FC assessment. The induction of apoptosis and DNA damage were studied using Annexin V-FITC/PI and DAPI. Results: Bacillus-derived antifungal variant AF4 was found to have structural features typical of lipopeptides. Microscopy imaging revealed that AF4 damages the surface of treated cells and results in membrane permeabilization, facilitating the uptake of the fluorescent dyes. A loss of membrane integrity was observed in cells treated with AF4 due to a decrease in DPH fluorescence and a dose-dependent increase in PI uptake. Cell damage was also determined from the log reduction of viable cells treated with AF4. AF4 treatment also caused elevated ROS levels, induced phosphatidylserine externalisation, late-stage apoptosis, and alterations to nuclear morphology revealed by DAPI fluorescence. Conclusion: Collectively, the mode of action studies revealed that AF4 acts primarily on the cell membrane of C. albicans and has the potential to act as an antifungal drug candidate.

A Cyclic Disulfide Diastereomer From Bioactive Fraction of Bruguiera gymnorhiza Shows Anti-Pseudomonas aeruginosa Activity.

Pseudomonas aeruginosa is an opportunistic pathogen that commonly causes hospital-acquired infection and is of great concern in immunocompromised patients. The quorum sensing (QS) mechanism of P. aeruginosa is well studied and known to be responsible for pathogenicity and virulence. The QS inhibitor derived from the natural product can be an important therapeutic agent for pathogen control. The present study reports the role of Bruguiera gymnorhiza purified fraction (BG138) in inhibiting virulence factor production, biofilm formation, quorum sensing molecules, and expression of QS-related genes of P. aeruginosa. Structural characterization of BG138 by high resolution mass spectrometry, Fourier transform infrared spectroscopy, 1D (1H and 13C NMR) and 2D NMR reveals that the fraction is a mixture of already known cyclic disulfide diastereomer, namely, brugierol and isobrugierol. The minimum inhibitory concentration (MIC) of BG138 against P. aeruginosa was 32 μg/ml. Biofilm formation was significantly reduced at sub-MIC concentrations of BG138. Scanning electron microscopy analysis reports the concentration-dependent biofilm inhibition and morphological changes of P. aeruginosa. Flow cytometry–based cell viability assay showed that P. aeruginosa cells exhibit increased propidium iodide uptake on treatment with 32 and 64 μg/ml of BG138. At sub-MIC concentrations, BG138 exhibited significant inhibition of virulence factors and reduced swimming and swarming motility of P. aeruginosa. Furthermore, the effect of BG138 on the expression of QS-related genes was investigated by qRT-PCR. Taken together, our study reports the isolation and structural characterization of bioactive fraction BG138 from B. gymnorhiza and its anti-biofilm, anti-virulence, anti-quorum sensing, and cell-damaging activities against P. aeruginosa.

Nitric Oxide Inhibits TRPV4‐mediated Hemichannel Opening in Porcine Nonpigmented Ciliary Epithelium

Previous studies showed the presence of connexin 43 (Cx43), connexin 50 (Cx50), and pannexin 1 (Panx1) in the nonpigmented ciliary epithelium (NPE) of the eye, as well as evidence of hemichannel (HC) activity. In the lens, mechanosensitive Transient Receptor Potential Vanilloid 4 (TRPV4)‐mediated calcium entry stimulates hemichannel activity. The aim here is to test for TRPV4 expression in the NPE, determine whether TRPV4 activation causes HC opening, and study the influence of nitric oxide.Immunohistochemistry was used to probe for TRPV4 in porcine ciliary body. Propidium iodide (PI) uptake and ATP release by primary cultured NPE was used to assess HC opening. PI and ATP are large molecules (MW 668 and 507, respectively) that pass through open hemichannels but not the plasma membrane. ATP in the bathing medium was measured by luciferase luminometry and PI uptake by fluorimetry.Abundant expression of TRPV4 was detected in the NPE cell layer of the ciliary body. Cultured NPE cells displayed an increase in PI uptake when exposed to a TRPV4 agonist GSK 1016790A (10 nM) or to hypoosmotic solution (200 mOsm) (Control 4.65 ± 0.24 vs GSK 10.26 ± 0.32 and Hypo 8.26 ± 0.42 fluorescence units per mg protein; n=6, p<0.001). PI uptake also was increased in NPE cells cultured on a flexible membrane and subjected to cyclic stretch for 1‐10 min (10%, 0.5 Hz), the peak response was at at 2 min (Control 1.5 ± 0.03 vs stretch 2.5 ± 0.10; n=6, p<0.001). The increase in PI uptake caused by hypoosmotic solution or by cyclic stretch was abolished by a selective TRPV4 antagonist HC 067047 (10 μM). Hyposmotic solution increased ATP release from 1.12 ± 0.24 to 2.81± 0.27 (n=12, p<0.001) while cyclic stretch increased ATP release from 0.20 ± 0.03 to 0.40 ± 0.03 pmoles/mg protein (n=6, p<0.001). ATP release responses caused by hypoosmotic solution and cyclic stretch were inhibited by HC 067047. Hypoosmotic solution‐induced ATP release was also inhibited by connexin inhibitory peptide, Gap‐27 (200 μM) and nitric oxide (NO) donors, sodium nitroprusside (SNP) and S‐nitroso‐N‐acetylpenicillamine (SNAP).TRPV4 channels are expressed by the NPE. The ability of HC 067047 and Gap‐27 to prevent ATP release and PI uptake points to TRPV4‐dependent opening of connexin HCs in NPE cells. Suppression of ATP release by SNP and SNAP indicates the hemichannel opening mechanism is inhibited by NO.

The OrganoPlate® as vessel-on-a-chip model to investigate increased microbubble-mediated vascular permeability

The vessel wall is an important barrier modulating drug delivery to the underlying diseased tissue. Oscillating microbubbles can be used to locally enhance vascular permeability and sonoporate cells. As the mechanism is not fully understood, our aim was to grow 3D human vessels-on-a-chip in the OrganoPlate® 40 and use this model to investigate the effect of αvβ3-targeted microbubble and different ultrasound pressures (2 MHz, 100–850 kPa peak negative pressure) and cycle lengths (10×10 or 10×1000 cycles) on vascular permeability and sonoporation. The vascular permeability of 122 microvessels in 14 different conditions was quantified by microscopy imaging using the leakage pattern of a 150 kDa FITC-dextran dye. Furthermore, sonoporation was assessed using propidium iodide (PI). Upon microbubble and ultrasound treatment, an increase in vascular permeability was observed. Higher pressures and longer cycle length treatment showed a significantly higher vascular permeability and significant increase in PI uptake compared to all control groups (sham, ultrasound only, microbubble only), suggesting a simultaneous increase in vascular permeability and sonoporation correlating with higher pressure and longer cycle insonifications. In conclusion, the vessel-on-chip model is a suitable model to investigate how insonification with different ultrasound settings affects the microbubble-mediated vascular permeability increase and sonoporation.

MO008NEPHROTOXIC MECHANISMS OF GADOLINIUM: IMPLICATIONS FOR THE USE OF GADOLINIUM-BASED CONSTRAST AGENTS

Abstract Background and Aims Gadolinium-based contrast agents are widely used for magnetic resonance imaging, and although they may be considered well tolerated at recommended dosing levels, recent evidences support the deposition of free gadolinium in the tissues, and its slow release into circulation, resulting in long-term toxicity, which is aggravated in renal patients. The kidney, as the major excretion organ of these agents, and particularly the proximal tubule, as a common location of xenobiotics’ bioaccumulation, may be key targets of gadolinium’s deleterious effects. This study aimed at unveiling the nephrotoxic potential and the underlying mechanisms of toxicity of gadolinium, using an in vitro model of normal human proximal tubular cells (HK-2 cell line). Method HK-2 cells were exposed for 24 h to a wide concentration range of gadolinium, in the form of trichloride hexahydrate, and cell viability was assessed to estimate the half maximal effective concentration (EC50) and the subtoxic concentration eliciting 1 % of cell death (EC01). These ECs were further used to determine the effects of gadolinium on the cell’s oxidative status, mitochondrial function, cell death mechanisms and lipid deposition, through variable colorimetric and fluorometric assays. Expression of the pro-inflammatory gene IL6 was also determined through quantitative PCR. Results Gadolinium induced cell death in a concentration-dependent manner, with estimated EC01 and EC50 of 3 and 340 µM, respectively. When compared to control cells, the subtoxic concentration showed no significant effects in terms of reactive oxygen and nitrogen species (ROS and RNS) production, total glutathione levels (tGSH) or total antioxidant status (TAS). On the other hand, the EC50 showed significant disruption of the oxidative status of the cell, with over 60 % depletion of tGSH cell contents (p < 0.01) and a significant decrease of TAS (p < 0.05). However, this effect was not accompanied by an increase, but rather by a significant decline in ROS and RNS production of about 50 % below control (p < 0.0001). At the EC50, but not at EC01, it was also observed the disturbance of the mitochondrial and energetic homeostasis, as showed by the increment of intracellular free calcium levels, hyperpolarization of the mitochondrial membrane, and decay of the ATP levels (p < 0.0001). Cell death induced by gadolinium was characterized by typical morphological changes of late apoptosis and necrosis, with a significant increase in propidium iodide uptake and lactate dehydrogenase leakage (p ≤ 0.0001) at EC50. The presence of neutral lipids-containing vesicles was observed in the cytoplasm of cells exposed to gadolinium using the fluorescent dye BODIPY, already noticeable at the subtoxic concentration. Cells exposed to gadolinium also showed increased expression of IL6, though this effect was only significant at the EC01 (p < 0.05). Conclusion Gadolinium showed marked cytotoxic potential at micromolar levels in HK-2 cells. This cytotoxicity was characterized by increased oxidative stress independent of ROS and RNS production and mitochondrial dysfunction followed by cell death via late apoptosis and necrosis. At a subtoxic concentration, gadolinium was also able to elicit the accumulation of lipidic vesicles within the cells’ cytoplasm, and to trigger a pro-inflammatory response. Although it is still unclear which amount of gadolinium is in fact released from the complexes commonly used as contrast agents, this study shows that gadolinium ion has direct nephrotoxic potential, with noteworthy manifestations at subtoxic concentrations. Acknowledgments This work was supported by Applied Molecular Biosciences Unit (UCIBIO), financed by national funds from FCT/MCTES (UIDB/04378/2020), by North Portugal Regional Coordination and Development Commission (CCDR-N)/NORTE2020/Portugal 2020 (Norte-01-0145-FEDER-000024).

Anti-Bacterial Properties of Cannabigerol Toward Streptococcus mutans.

Streptococcus mutans (S. mutans) is a gram-positive facultatively anaerobic bacterium and the most common pathogen associated with tooth caries. The organism is acid tolerant and can undergo physiological adaptation to function effectively in acid environments such as carious dental plaque. Some cannabinoids have been found to have potent anti-microbial activity against gram-positive bacteria. One of these is the non-psychoactive, minor phytocannabinoid Cannabigerol (CBG). Here we show that CBG exhibits anti-bacterial activities against S. mutans. CBG halts the proliferation of planktonic growing S. mutans, which is affected by the initial cell density. High-resolution scanning electron microscopy showed that the CBG-treated bacteria become swollen with altered membrane structures. Transmission electron microscopy provided data showing that CBG treatment leads to intracellular accumulation of membrane structures. Nile red, DiOC2(3) and laurdan staining demonstrated that CBG alters the membrane properties, induces membrane hyperpolarization, and decreases the membrane fluidity. CBG-treated bacteria showed increased propidium iodide uptake and reduced calcein AM staining, suggesting that CBG increases the membrane permeability and reduces the metabolic activity. Furthermore, CBG prevented the drop in pH caused by the bacteria. In summary, we present here data showing the mechanisms by which CBG exerts its anti-bacterial effect against S. mutans.

Neuroprotective effects of microglial P2Y1 receptors against ischemic neuronal injury

Extracellular ATP, which is released from damaged cells after ischemia, activates P2 receptors. P2Y1 receptors (P2Y1R) have received considerable attention, especially in astrocytes, because their activation plays a central role in the regulation of neuron-to-glia communication. However, the functions or even existence of P2Y1R in microglia remain unknown, despite the fact that many microglial P2 receptors are involved in several brain diseases. Herein, we demonstrate the presence and functional capability of microglial P2Y1R to provide neuroprotective effects following ischemic stress. Cerebral ischemia resulted in increased microglial P2Y1R expression. The number of injured hippocampal neurons was significantly higher in P2Y1 R knockout (KO) mice than wildtype mice after forebrain ischemia. Propidium iodide (PI) uptake, a marker for dying cells, was significantly higher in P2Y1R KO hippocampal slices compared with wildtype hippocampal slices at 48 h after 40-min oxygen–glucose deprivation (OGD). Furthermore, increased PI uptake following OGD was rescued by ectopic overexpression of P2Y1R in microglia. In summary, these data suggest that microglial P2Y1R mediate neuroprotective effects against ischemic stress and OGD insult.

A method for objectively quantifying propidium iodide exclusion in organotypic hippocampal slice cultures

BackgroundOrganotypic hippocampal slice cultures (OHSCs) are an attractive in vitro model to examine mechanisms of neuronal injury, because the normal hippocampal architecture, function and cellular diversity are mostly preserved. The effects of exposure to excitotoxins such as N-methyl-d-aspartate (NMDA) on cell viability can be determined by propidium iodide (PI) staining. New methodWe describe a simple method to objectively quantify cell death in NMDA exposed slice cultures using PI that provides a standardized means of quantifying cell death in hippocampal subfields without the need to induce maximal cell death in each slice. The method employs separation of subfields using simple landmarks and densitometric quantification of PI intensity in 10 template-oriented counting fields. ResultsWe show that exposure to increasing concentrations of NMDA results in a dose-dependent increase in PI uptake. Additionally, our method facilitates the comparison of cell death in different hippocampal subfields, such as dentate gyrus, CA1 and CA3. Our results show marked differences of PI uptake in the hippocampal regions with the CA1 area being most sensitive to NMDA-induced injury. Comparison with existing method(s)The method provides a standardized format for quantifying PI exclusion in OHSCs that can be applied to cultures of differing shapes and sizes, permits comparisons between hippocampal subfields and does not require induction of maximal cell death. ConclusionThe method of quantifying PI uptake described herein allows for an objective, quantitative and reproducible analysis and comparison of cell death in distinct regions of OHSCs.

Toxicity of amyloid beta 1-40 and 1-42 on SH-SY5Y cell line

Objectives: Amyloid beta plaques are primary hallmark of Alzheimer’s disease, which is characterized by specific neurodegeneration. Amyloid beta peptide – the main plaque component- was shown to be neurotoxic in animal models, primary neuronal cultures and immortalized cell lines. However, the results are often controversial and there is no good human cell line model for evaluation of the toxicity of amyloid peptides. Here we studied the effect of amyloid beta 1-40 and 1-42 on undifferentiated and differentiated human neuroblastoma cell line SH-SY5Y. Results: Undifferentiated cell culture was too diverse and unstable to reveal a toxic effect of amyloid beta peptides quantitatively. Differentiated cells established more neuron-like phenotype and were more identical and stable in culture suggesting potential susceptibility to amyloid beta as a neurotoxic agent. Amyloid peptides are prone to form different aggregates with diverse toxic properties, in current study, monomeric amyloid beta 1-40 and 1-42 were applied to the cells. Viability test WST-1 and propidium iodide (PI) uptake tests showed that undifferentiated cells are not susceptible to amyloid beta, however, differentiated cells showed reduced viability and increased PI uptake in case of amyloid beta 1-42, but not in case of amyloid beta 1-40. Conclusions: Current study revealed that amyloid beta has no remarkably toxic effect on undifferentiated SH-SY5Y cell line whereas viability of the neuron-like differentiated cell culture is significantly decreased by the amyloid beta 1-42 peptide that is known to form spontaneously toxic aggregates.

Concentration-Dependent Bimodal Effect of Specific 18 kDa Translocator Protein (TSPO) Ligands on Cell Death Processes Induced by Ammonium Chloride: Potential Implications for Neuropathological Effects Due to Hyperammonemia

The role of the 18-kDa Translocator Protein (TSPO) in cell death induced by NH4Cl (1-50 mM) for 24-72 hours to human glioblastoma U118MG cells was investigated. Cell death was already observed after 48 hours of treatment with NH4Cl at 5 mM. Dose and time-responses curves indicated that 15 mM of NH4Cl applied for 72 hours was the optimal condition for our viability assays. For example, 72 hours of 15 mM of NH4Cl caused a 50.3% increase in propidium iodide uptake, and lactate dehydrogenase release was 41.2% of the positive control, indicating significant increases in cell death. Furthermore, compared to vehicle control, these experimental conditions resulted in a significant decrease of 44.9% of the mitochondrial activity, a 62.3% increase in incidence of collapse of mitochondrial membrane potential, and an increase of 49.0% of cardiolipin peroxidation. In addition, a significant 4.3 fold increase in the maximal binding capacity (Bmax) of TSPO was found in NH4Cl-exposed cells. Surprisingly, western blot analysis and real-time PCR did not demonstrate changes in TSPO expression. We also found that neither NH4Cl nor glutamine (a metabolic product of enhanced NH4Cl levels) inhibited binding of the TSPO ligand [(3)H]PK 11195. Interestingly, we observed a bimodal effect of the TSPO ligands PK 11195, Ro5-4864, and FGIN-1-27 on the toxicity of NH4Cl; such that 1-100 nM concentrations of TSPO ligands were protective, while concentrations above 1 μM enhanced NH4Cl-induced cell death processes. In conclusion, TSPO takes part in a bimodal way in the lethal effects induced by NH4Cl in glial type cells.

Photodynamic antibacterial effect of graphene quantum dots

Synthesis of new antibacterial agents is becoming increasingly important in light of the emerging antibiotic resistance. In the present study we report that electrochemically produced graphene quantum dots (GQD), a new class of carbon nanoparticles, generate reactive oxygen species when photoexcited (470 nm, 1 W), and kill two strains of pathogenic bacteria, methicillin-resistant Staphylococcus aureus and Escherichia coli. Bacterial killing was demonstrated by the reduction in number of bacterial colonies in a standard plate count method, the increase in propidium iodide uptake confirming the cell membrane damage, as well as by morphological defects visualized by atomic force microscopy. The induction of oxidative stress in bacteria exposed to photoexcited GQD was confirmed by staining with a redox-sensitive fluorochrome dihydrorhodamine 123. Neither GQD nor light exposure alone were able to cause oxidative stress and reduce the viability of bacteria. Importantly, mouse spleen cells were markedly less sensitive in the same experimental conditions, thus indicating a fairly selective antibacterial photodynamic action of GQD.

Membrane-damaging potential of natural L-(−)-usnic acid in Staphylococcus aureus

The purpose of this investigation was to try to understand the antibacterial mechanism of L-(-)-usnic acid isolated for the first time from fruticose lichen Usnea subfloridana using clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA). The minimum inhibitory concentration (MIC) of L-(-)-usnic acid against the clinical isolates of MRSA and reference strain S. aureus MTCC-96 (SA-96) was in the range 25-50 μg/ml. Treatment of both reference and clinical strains (MRSA-ST 2071) with four-fold MIC concentrations (100-200 μg/ml) of L-(-)-usnic acid reduced the viability of cells without damaging the cell wall. However, the loss of 260 nm absorbing material and increase in propidium iodide uptake was observed in both of the strains. Similarly, a combined effect of L-(-)-usnic acid (25-50 μg/ml) and 7.5 % NaCl resulted in a reduced number of viable cells within 24 h in comparison to the control. These observations clearly indicate that L-(-)-usnic acid exerts its action by disruption of the bacterial membrane. Further, in vivo efficacy showed that L-(-)-usnic acid significantly (p < 0.001) lowered the microbial load of spleen at doses ranging from 1 to 5 mg/kg. Further, toxicity studies in infected mice at doses 20 times higher than the efficacious dose indicated L-(-)usnic acid to be safe. Paradoxically, L-(-)usnic acid exhibited changes in serum triglycerides, alkaline phosphatase (ALKP) and liver organ weight in the healthy mice administered with only 25 mg/kg body weight. The results obtained in this study showed that natural L-(-)-usnic acid exerts its antibacterial activity against MRSA by disruption of the cell membrane. Further, the natural L-(-)-usnic acid was found to be safe up to 100 mg/kg body weight, thereby, making it a probable candidate for treating S. aureus infections.

TRPV4 in porcine lens epithelium regulates hemichannel-mediated ATP release and Na-K-ATPase activity

In several tissues, transient receptor potential vanilloid 4 (TRPV4) channels are involved in the response to hyposmotic challenge. Here we report TRPV4 protein in porcine lens epithelium and show that TRPV4 activation is an important step in the response of the lens to hyposmotic stress. Hyposmotic solution (200 mosM) elicited ATP release from intact lenses and TRPV4 antagonists HC 067047 and RN 1734 prevented the release. In isosmotic solution, the TRPV4 agonist GSK1016790A (GSK) elicited ATP release. When propidium iodide (PI) (MW 668) was present in the bathing medium, GSK and hyposmotic solution both increased PI entry into the epithelium of intact lenses. Increased PI uptake and ATP release in response to GSK and hyposmotic solution were abolished by a mixture of agents that block connexin and pannexin hemichannels, 18α-glycyrrhetinic acid and probenecid. Increased Na-K-ATPase activity occurred in the epithelium of lenses exposed to GSK and 18α-glycyrrhetinic acid + probenecid prevented the response. Hyposmotic solution caused activation of Src family kinase and increased Na-K-ATPase activity in the lens epithelium and TRPV4 antagonists prevented the response. Ionomycin, which is known to increase cytoplasmic calcium, elicited ATP release, the magnitude of which was no greater when lenses were exposed simultaneously to ionomycin and hyposmotic solution. Ionomycin-induced ATP release was significantly reduced in calcium-free medium. TRPV4-mediated calcium entry was examined in Fura-2-loaded cultured lens epithelium. Hyposmotic solution and GSK both increased cytoplasmic calcium that was prevented by TRPV4 antagonists. The cytoplasmic calcium rise in response to hyposmotic solution or GSK was abolished when calcium was removed from the bathing solution. The findings are consistent with hyposmotic shock-induced TRPV4 channel activation which triggers hemichannel-mediated ATP release. The results point to TRPV4-mediated calcium entry that causes a cytoplasmic calcium increase which is an essential early step in the mechanism used by the lens to sense and respond to hyposmotic stress.

Effects of Chemotherapeutics on Organotypic Corticostriatal Slice Cultures Identified by A Panel of Fluorescent and Immunohistochemical Markers

Effects of chemotherapeutics on glioma cell lines and spheroids are usually investigated without evaluating the effects of chemotherapeutics on normal brain tissue. To perform such investigations, the aim of this study was to establish a panel of markers for detection of general cell death and more specific neuronal and glial degeneration induced by chemotherapeutics in organotypic rat corticostriatal slice cultures. The slice cultures were exposed to the alkylating agents temozolomide (TMZ) and nimustine (ACNU), the tyrosine kinase inhibitor imatinib mesylate (IM) and the microtubule-destabilizing agent vincristine (VCR). Densitometric measurements of uptake of the fluorescent dye propidium iodide (PI) were used for quantifying cellular degeneration. Moreover, paraffin sections were hematoxylin eosine stained and immunostained for the neuronal marker microtubule-associated protein 2 (MAP2), the astroglial marker glial fibrillary acidic protein (GFAP), and the oligodendroglial marker p25α. The results showed that the supposed clinically relevant drug concentrations were non-toxic. However, a time dependent increase in PI uptake was observed for high drug concentrations, except for TMZ, where no toxicity was observed. Corresponding immunostaining showed loss of MAP2 and increased expression of GFAP and p25α for cultures exposed to 1,000nM VCR. Cultures exposed to high concentrations of ACNU and IM disintegrated, leaving no tissue for histology. In conclusion, corticostriatal slice cultures and the established panel of markers represent an excellent tool for detecting toxicity induced by chemotherapeutics. Toxicity was not detected at clinical concentrations, but high concentrations with toxic effects were identified suggesting that some of the earlier identified anti-cancer effects are general cytotoxic effects and not specific anti-cancer effects.

Propidium iodide staining method for testing the cytotoxicity of 2,4,6-trichlorophenol and perfluorooctane sulfonate at low concentrations with Vero cells

In this article, the feasibility of propidium iodide (PI) staining based on cell membrane damage as a sensitive and quantitative method to test the cytotoxicity of typical lipophilic compounds including 2,4,6-trichlorophenol (TCP) and perfluorooctane sulfonate (PFOS) was examined. The sensitivity of PI staining was compared to that of the methylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT) assay. We found a good correlation between PI uptake and increasing concentrations of TCP (10–50 μM) and PFOS (20–50 μM) at lower concentrations than those between growth inhibition ratio determined by the MTT assay and increasing concentrations of TCP (150–400 μM) and PFOS (100–500 μM). These findings indicated that the PI staining was more sensitive than the MTT assay. Furthermore, both the amount of PI uptake and the ratio of morphologically changed cells increased with increasing TCP concentrations, suggesting that PI staining may be a suitable quantitative substitute for the original method based on the observation of morphological changes in Vero cells. Our data also suggest that it is possible that the cell membrane damage that resulted in increased PI uptake may be due to variations in the phospholipid and protein content of the membrane, which are affected by interactions between the lipophilic compounds and components of the cell membrane.

Abstract 568: Effects of irradiation on glioblastoma spheroids cultured in stem cell medium

Abstract Radiotherapy is an important part of the treatment of glioblastomas – the most frequent and malignant primary brain tumor. However, the current treatment remains palliative and new treatments are urgently needed. Since a subpopulation of cells – cancer stem cells – is believed to be particularly resistant to the current treatment and is most likely the cause of tumor recurrence, the use of models preserving cancer stem cell features is of increasing importance. Recent reports have suggested that glioblastoma spheroids derived from short term cultures in stem cell medium preserve the pheno- and genotype better than when cultured in serum containing medium. With this background, the aim of this study was to investigate the response of irradiation of such spheroids as well as spheroids implanted into organotypic corticostriatal slice cultures in order to have a model preserving the invasive feature of glioblastomas. The response to irradiation (2, 5, 10 and 50 Gy) was investigated in spheroids from a short term culture with cancer stem cell-like properties established in our laboratory (C76) and the U87 glioblastoma cell line for comparison. Cell death was measured by uptake of propidium iodide (PI) and release of lactate dehydrogenase (LDH) and moreover, expression of p53 and the proliferation marker (MIB-1) was assessed by immunohistochemical staining. The formation of secondary spheroids after irradiation was evaluated using a limiting dilution assay as well as a spheroid proliferation assay at non-clonal density. Finally, fluorescently labeled C76 spheroids implanted into slice cultures were irradiated and the response on invasion was evaluated by confocal microscopy and anti-human vimentin immunohistochemical staining. For both C76 and U87 spheroids, the results showed a minor increase in PI uptake with increasing irradiation dose but no increase in LDH release. The expression of p53 also increased, whereas MIB-1 was reduced. The spheroid formation capacity in the limiting dilution assay was higher for C76 than U87 but decreased for both C76 and U87 with increasing irradiation dose along with a decrease in the number and size of secondary formed spheroids in the spheroid proliferation assay. The spheroid formation capacity was clearly best preserved for C76, but otherwise the results did not show major differences for C76 and U87. Using the invasion assay, the invasion of C76 spheroids into brain tissue was not affected by irradiation. In conclusion, irradiation of glioblastoma spheroids derived from short term cultures with cancer stem cell-like properties is feasible and easy to use as an in vitro model. The results might suggest that irradiation also affects tumor cells with a more stem-like phenotype although the effects on the unique tumor stem cells need to be further investigated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 568. doi:10.1158/1538-7445.AM2011-568

Sex Differences in Caffeine Neurotoxicity Following Chronic Ethanol Exposure and Withdrawal

Caffeine is a central nervous system stimulant that produces its primary effects via antagonism of the A(1) and A(2A) adenosine receptor subtypes. Previous work demonstrated a sex difference in neurotoxicity produced by specific adenosine A(1) receptor antagonism during ethanol withdrawal (EWD) in vitro that was attributable to effects downstream of A(1) receptors at NMDA receptors. The current studies were designed to examine the effect of non-specific adenosine receptor antagonism with caffeine during ethanol withdrawal on hippocampal toxicity in cultures derived from male and female rats. At 5 days in vitro (DIV), half of the male and female organotypic hippocampal slice cultures were exposed to 50 mM ethanol (EtOH) in culture media for 10 days before exposure to caffeine (5, 20 and 100 microM) for the duration of a 24 h EWD period. In keeping with this timeline, the remaining ethanol-naïve cultures were given media changes at 10 and 15 DIV and exposed to caffeine (5, 20 and 100 microM) for 24 h at 15 DIV. Cytotoxicity was assessed by fluorescent microscopy and quantification of propidium iodide (PI) uptake in the pyramidal cell layers of the CA1 and CA3 regions and the granule cell layer of the dentate gyrus (DG). A two-way (sex x treatment) ANOVA was conducted within each hippocampal region. Twenty-four-hour withdrawal from 10-day exposure to 50 mM ethanol did not produce increased PI uptake in any hippocampal region. Caffeine exposure (5, 20 and 100 microM) in ethanol-naïve cultures did not produce toxicity in the DG or CA1 region, but 20 microM caffeine produced modest toxicity in the CA3 region. Exposure to 20 microM caffeine during EWD produced cytotoxicity in all hippocampal regions, though toxicity was sex-dependent in the DG and CA1 region. In the DG, both 5 and 20 microM caffeine produced significantly greater PI uptake in ethanol-exposed female cultures compared to ethanol-naïve female cultures and all male cultures. Similarly, 20 microM caffeine caused markedly greater toxicity in female cultures as compared to male cultures in the CA1 region. Twenty-four-hour exposure to caffeine during EWD produced significant toxicity in the pyramidal cell layer of the CA3 region in male and female cultures, though toxicity in the granule cell layer of the DG and pyramidal cell layer of the CA1 region was observed only in female cultures. Greater sensitivity of the female slice cultures to toxicity upon caffeine exposure after prolonged ethanol exposure is consistent with previous studies of effects of a specific A(1) receptor antagonism during EWD on toxicity and indicate that this effect is independent of the hormonal milieu. Together, these data suggest that the A(1) receptor subtype is predominant in mediating caffeine's neurotoxic effects during EWD. These findings demonstrate the importance of considering gender/sex when examining neuroadaptive changes in response to ethanol exposure and withdrawal.