Simple discrimination of sub-cycling cells by propidium iodide flow cytometric assay in Jurkat cell samples with extensive DNA fragmentation.

Human leukemia Jurkat T cells were analyzed for apoptosis and cell cycle by flow cytometry, using the Annexin V/propidium iodide (PI) standard assay, and a simple PI staining in Triton X-100/digitonin-enriched PI/RNase buffer, respectively. Cells treated with doxorubicin or menadione displayed a very strong correlation between the apoptotic cell fraction measured by the Annexin V/PI assay, and the weight of a secondary cell population that emerged on the forward scatter (FS)/PI plot, as well as on the side scatter (SS)/PI and FL1/PI plots generated from parallel cell cycle recordings. In both cases, the Pearson correlation coefficients were >0.99. In cell cycle determinations, PI fluorescence was detected on FL3 (620/30 nm), and control samples exhibited the expected linear dependence of FL3 on FL1 (525/40 nm) signals. However, increasing doses of doxorubicin or menadione generated a growing subpopulation of cells displaying a definite right-shift on the FS/FL3, SS/FL3 and FL1/FL3 plots, as well as decreased PI fluorescence, indicative of ongoing fragmentation and loss of nuclear DNA. By gating on these events, the resulting fraction of presumably sub-cycling cells (i.e. cells with cleaved DNA, counting sub-G0/G1, sub-S and sub-G2/M cells altogether) was closely similar to the apoptotic rate assessed by Annexin V/PI labeling. Taken together, these findings suggest a possible way to recognize the entire population of cells undergoing apoptotic DNA cleavage and simultaneously determine the cell cycle distribution of non-apoptotic cells in PI-labeled cell samples with various degrees of DNA fragmentation, using a simple and reproducible multiparametric analysis of flow cytometric recordings.

Cellular determinants involving mitochondrial dysfunction, oxidative stress and apoptosis correlate with the synergic cytotoxicity of epigallocatechin-3-gallate and menadione in human leukemia Jurkat T cells.

We have investigated the growth-suppressive action of epigallocatechin-3-gallate (EGCG) on human leukemia Jurkat T cells. Results show a strong correlation between the dose-dependent reduction of clonogenic survival following acute EGCG treatments and the EGCG-induced decline of the mitochondrial level of Ca(2+). The cell killing ability of EGCG was synergistically enhanced by menadione. In addition, the cytotoxic effect of EGCG applied alone or in combination with menadione was accompanied by apoptosis induction. We also observed that in acute treatments EGCG displays strong antioxidant properties in the intracellular milieu, but concurrently triggers some oxidative stress generating mechanisms that can fully develop on a longer timescale. In parallel, EGCG dose-dependently induced mitochondrial depolarization during exposure, but this condition was subsequently reversed to a persistent hyperpolarized mitochondrial state that was dependent on the activity of respiratory Complex I. Fluorimetric measurements suggest that EGCG is a mitochondrial Complex III inhibitor and indicate that EGCG evokes a specific cellular fluorescence with emission at 400nm and two main excitation bands (at 330nm and 350nm) that may originate from a mitochondrial supercomplex containing dimeric Complex III and dimeric ATP-synthase, and therefore could provide a valuable means to characterize the functional properties of the respiratory chain.

Ultra-weak delayed luminescence in cancer research: a review of the results by the ARETUSA equipment.

The study of the photoinduced ultraweak photon emission in the optical wavelength range, namely the Delayed Luminescence, from human cells and tissues has an increasingly growing interest in view of its possible application in optical biopsy. Due to the low level, dedicated experimental set-up are necessary to reveal such photoluminescence signal. The paper reviews the results obtained in the field of cancer research, by using the experimental equipment for fast ultraweak luminescence analysis ARETUSA developed at the National Southern Laboratories of the National Nuclear Physics Institute (LNS-INFN), in Catania, Italy. Delayed Luminescence signals from normal and cancer cells are compared and the relationship between Delayed Luminescence and apoptosis is investigated.

Novel insights into the antiproliferative effects and synergism of quercetin and menadione in human leukemia Jurkat T cells.

The flavonoid quercetin and menadione (vitamin K3) are known as potent apoptogens in human leukemia Jurkat T cells. We explored some underlying mechanisms and the potential relevance of the combination quercetin-menadione for clinical applications. In acute treatments, quercetin manifested a strong antioxidant character, but induced a transient loss of Δψm, likely mediated by opening of the mitochondrial permeability transition pore. After removal of quercetin, persistent mitochondrial hyperpolarization was generated via stimulation of respiratory Complex I. In contrast, menadione-induced Δψm dissipation was only partially and transiently reversed after menadione removal. Results indicate that Ca(2+) release is a necessary event in quercetin-induced cell death and that the survival response to quercetin is delineated within 1h from exposure. Depending on dose, the two agents exhibited either antagonistic or synergistic effects in reducing clonogenicity of Jurkat cells. 24-h combinatorial regimens at equimolar concentrations of 10-15 µM, which are compatible with a clinically achievable (and safe) scheme, reduced cell viability at efficient rates. Altogether, these findings support the idea that the combination quercetin-menadione could improve the outcome of conventional leukemia therapies, and warrant the utility of additional studies to investigate the therapeutic effects of this combination in different cellular or animal models for leukemia.

Polymorphism of hen egg white lysozyme amyloid fibrils influences the cytotoxicity in LLC-PK1 epithelial kidney cells.

The polymorphism of amyloid fibrils is potentially crucial as it may underlie the natural variability of amyloid diseases and could be important in developing a fuller understanding of the molecular basis of protein deposition disorders. This study examines morphological differences in lysozyme fibrils and the implications of these differences in terms of cytotoxicity. The structural characteristics of amyloid fibrils formed under two different experimental conditions (acidic and neutral) were evaluated using spectroscopic methods, atomic force microscopy and image analysis. Growth curves and apoptotic/necrotic assays were used to determine the cytotoxic effect of fibrils on the LLC-PK1 renal cells. The results reveal that both types of mature lysozyme amyloid fibrils are actively involved in the cytotoxic process, however each exhibit different levels of cytotoxicity. Fibrils formed at acidic pH affect cell growth in a dose-dependent manner, but a threshold-dependent inhibition of cell growth was observed in the case of lysozyme fibrils prepared at neutral pH. Experiments examining the mechanism of the cell death suggest that both types of mature lysozyme fibrils trigger late apoptosis/necrosis at different fibril concentrations. Our findings clearly indicate that the intrinsic differences between amyloid fibrils due to their polymorphism result in different degrees of cytotoxicity.

Epigallocatechin 3-O-gallate induces 67 kDa laminin receptor-mediated cell death accompanied by downregulation of ErbB proteins and altered lipid raft clustering in mammary and epidermoid carcinoma cells.

Since the administration of synthetic medicines is associated with drug resistance and undesired side effects, utilization of natural compounds could be an alternative and complementary modality to inhibit or prevent the development of tumors. Epigallocatechin 3-O-gallate (EGCG, 1), the major flavan component of green tea, and genistein (2), a soy isoflavonoid, are known to have chemopreventive and chemotherapeutic effects against cancer. This study demonstrated that both flavonoids inhibit cell proliferation, an effect enhanced under serum-free conditions. Compound 1, but not 2, induced downregulation of ErbB1 and ErbB2 in mammary and epidermoid carcinoma cells, and its inhibitory effect on cell viability was mediated by the 67 kDa laminin receptor (67LR). While 1 was superior in inducing cell death, 2 was more efficient in arresting the tumor cells in the G2/M phase. Furthermore, number and brightness analysis revealed that 1 decreased the homoclustering of a lipid raft marker, glycosylphosphatidylinositol-anchored GFP, and it also reduced the co-localization between lipid rafts and 67LR. The main conclusion made is that the primary target of 1 may be the lipid raft component of the plasma membrane followed by secondary changes in the expression of ErbB proteins. Compound 2, on the other hand, must have other unidentified targets.

Mitochondrial respiratory complex I probed by delayed luminescence spectroscopy.

The role of mitochondrial complex I in ultraweak photon-induced delayed photon emission [delayed luminescence (DL)] of human leukemia Jurkat T cells was probed by using complex I targeting agents like rotenone, menadione, and quercetin. Rotenone, a complex I-specific inhibitor, dose-dependently increased the mitochondrial level of reduced nicotinamide adenine dinucleotide (NADH), decreased clonogenic survival, and induced apoptosis. A strong correlation was found between the mitochondrial levels of NADH and oxidized flavin mononucleotide (FMNox) in rotenone-, menadione- and quercetin-treated cells. Rotenone enhanced DL dose-dependently, whereas quercetin and menadione inhibited DL as well as NADH or FMNox. Collectively, the data suggest that DL of Jurkat cells originates mainly from mitochondrial complex I, which functions predominantly as a dimer and less frequently as a tetramer. In individual monomers, both pairs of pyridine nucleotide (NADH/reduced nicotinamide adenine dinucleotide phosphate) sites and flavin (FMN-a/FMN-b) sites appear to bind cooperatively their specific ligands. Enhancement of delayed red-light emission by rotenone suggests that the mean time for one-electron reduction of ubiquinone or FMN-a by the terminal Fe/S center (N2) is 20 or 284 µs, respectively. All these findings suggest that DL spectroscopy could be used as a reliable, sensitive, and robust technique to probe electron flow within complex I in situ.

Quercetin as a fluorescent probe for the ryanodine receptor activity in Jurkat cells.

Because channels of intracellular organelles are not directly accessible to the patch-clamp technique, the activity (open probability) of intracellular ion channels in intact cells has so far eluded direct examination. Here, we present strong evidence that the ratio F380/F440 of the quercetin-specific cellular fluorescence emitted at 540 nm upon excitation at 380/440 nm reflects the open probability of an endoplasmic reticulum Ca(2+) release channel, the ryanodine receptor (RyR), in both intact and permeabilized Jurkat cells. The time course of the Ca(2+) release signal induced by high levels of quercetin in intact cells and that of F380/F440 were strongly correlated. The RyR specific inhibitor, ryanodine, the RyR type 3 and 1 but not type 2 specific inhibitor, dantrolene, as well as the non-specific RyR inhibitor, ruthenium red, depressed consistently the quercetin-induced Ca(2+) transient. Confocal microscopy confirmed that the dual fluorescent signal emitted by quercetin colocalizes with the endoplasmic reticulum, not the mitochondria. A novel regulatory mechanism was identified whereby RyR activity under physiological conditions is partially suppressed (hindered channel), whereas the channel becomes nearly fully activated after exposure to millimolar concentrations of bulk cytosolic Ca(2+) and subsequent chelation of Ca(2+) (rectified channel). Upon rectification, the dependence of F380/F440 on the cytosolic Ca(2+) concentration was remarkably similar to that of the open probability of the RyR type 3, not 1 or 2, reported from bilayer experiments. So, quercetin appears to be a semi-specific fluorescent probe for the activity of ryanodine receptors, which in our Jurkat (clone E6.1) cell preparations probably reports the type 3 RyR activity.

Detailed analysis of apoptosis and delayed luminescence of human leukemia Jurkat T cells after proton irradiation and treatments with oxidant agents and flavonoids.

Following previous work, we investigated in more detail the relationship between apoptosis and delayed luminescence (DL) in human leukemia Jurkat T cells under a wide variety of treatments. We used menadione and hydrogen peroxide to induce oxidative stress and two flavonoids, quercetin, and epigallocatechin gallate, applied alone or in combination with menadione or H(2)O(2). 62 MeV proton beams were used to irradiate cells under a uniform dose of 2 or 10 Gy, respectively. We assessed apoptosis, cell cycle distributions, and DL. Menadione, H(2)O(2) and quercetin were potent inducers of apoptosis and DL inhibitors. Quercetin decreased clonogenic survival and the NAD(P)H level in a dose-dependent manner. Proton irradiation with 2 Gy but not 10 Gy increased the apoptotic rate. However, both doses induced a substantial G(2)/M arrest. Quercetin reduced apoptosis and prolonged the G(2)/M arrest induced by radiation. DL spectroscopy indicated that proton irradiation disrupted the electron flow within Complex I of the mitochondrial respiratory chain, thus explaining the massive necrosis induced by 10 Gy of protons and also suggested an equivalent action of menadione and quercetin at the level of the Fe/S center N2, which may be mediated by their binding to a common site within Complex I, probably the rotenone-binding site.

Diffusion-convection effects on drug distribution at the cell membrane level in a patch-clamp setup.

We present a model-based method for estimating the effective concentration of the active drug applied by a pressure pulse to an individual cell in a patch-clamp setup, which could be of practical use in the analysis of ligand-induced whole-cell currents recorded in patch-clamp experiments. Our modelling results outline several important factors which may be involved in the high variability of the electric response of the cells, and indicate that with a pressure pulse duration of 1s and diameter of the perfusion tip of 600 µm, elevated amounts of drug can accumulate locally between the pipette tip and the cell. Hence, the effective agonist concentration at the cell membrane level can be consistently higher than the initial concentration inside the perfusion tubes. We performed finite-difference and finite-element simulations to investigate the diffusion/convection effects on the agonist distribution on the cell membrane. Our model can explain the delay between the commencement of acetylcholine application and the onset of the whole-cell current that we recorded on human rhabdomyosarcoma TE671 cells, and reproduce quantitatively the decrease of signal latency with the concentration of agonist in the pipette. Results also show that not only the geometry of the bath chamber and pipette tip, but also the transport parameters of the diffusive and convective phenomena in the bath solution are determinant for the amplitude and kinetics of the recorded currents and have to be accounted for when analyzing patch-clamp data.

Effects of menadione, hydrogen peroxide, and quercetin on apoptosis and delayed luminescence of human leukemia Jurkat T-cells.

Menadione (MD) is an effective cytotoxic drug able to produce intracellularly large amounts of superoxide anion. Quercetin (QC), a widely distributed bioflavonoid, can exert both antioxidant and pro-oxidant effects and is known to specifically inhibit cell proliferation and induce apoptosis in different cancer cell types. We have investigated the relation between delayed luminescence (DL) induced by UV-laser excitation and the effects of MD, hydrogen peroxide, and QC on apoptosis and cell cycle in human leukemia Jurkat T-cells. Treatments with 500 µM H2O2 and 250 µM MD for 20 min produced 66.0 ± 4.9 and 46.4 ± 8.6% apoptotic cell fractions, respectively. Long-term (24 h) pre-exposure to 5 µM, but not 0.5 µM QC enhanced apoptosis induced by MD, whereas short-term (1 h) pre-incubation with 10 µM QC offered 50% protection against H2O2-induced apoptosis, but potentiated apoptosis induced by MD. Since physiological levels of QC in the blood are normally less than 10 µM, these data can provide relevant information regarding the benefits of flavonoid-combined treatments of leukemia. All the three drugs exerted significant effects on DL. Our data are consistent with (1) the involvement of Complex I of the mitochondrial respiratory chain as an important source of delayed light emission on the 10 µs-10 ms scale, (2) the ability of superoxide anions to quench DL on the 100 µs-10 ms scale, probably via inhibition of reverse electron transfer at the Fe/S centers in Complex I, and (3) the relative insensitivity of DL to intracellular OH• and H2O2 levels.