Jacek Grebowski

Fullerenols as a New Therapeutic Approach in Nanomedicine

Recently, much attention has been paid to the bioactive properties of water-soluble fullerene derivatives: fullerenols, with emphasis on their pro- and antioxidative properties. Due to their hydrophilic properties and the ability to scavenge free radicals, fullerenols may, in the future, provide a serious alternative to the currently used pharmacological methods in chemotherapy, treatment of neurodegenerative diseases, and radiobiology. Some of the most widely used drugs in chemotherapy are anthracycline antibiotics. Anthracycline therapy, in spite of its effective antitumor activity, induces systemic oxidative stress, which interferes with the effectiveness of the treatment and results in serious side effects. Fullerenols may counteract the harmful effects of anthracyclines by scavenging free radicals and thereby improve the effects of chemotherapy. Additionally, due to the hollow spherical shape, fullerenols may be used as drug carriers. Moreover, because of the existence of the currently ineffective ways for neurodegenerative diseases treatment, alternative compounds, which could prevent the negative effects of oxidative stress in the brain, are still sought. In the search of alternative methods of treatment and diagnosis, todays science is increasingly reaching for tools in the field of nanomedicine, for example, fullerenes and their water-soluble derivatives, which is addressed in the present paper.

Membrane fluidity and activity of membrane ATPases in human erythrocytes under the influence of polyhydroxylated fullerene.

The influence of fullerenol on the activities of human erythrocyte membrane ATPases and the fluidity of the plasma membrane as well as the possibility of fullerenol incorporation into the plasma membrane were investigated. Fullerenol at concentrations up to 150 μg/mL induced statistically significant decreases in the anisotropy of 1-anilino-8-naphthalene sulfonate (ANS) (14%), N,N,N-trimethyl-4-(6-phenyl-1,3,5,-hexatrien-1-yl)phenylammonium p-toluenesulfonate (TMA-DPH) (7.5%) and 1,6-diphenyl-1,3,5-hexatriene (DPH) (9.5%) after a 1-hour incubation at 37°C. The effect disappeared for ANS and TMA-DPH, but not for DPH, after washing out the fullerenol. Incubation of erythrocyte membranes with fullerenol led to decreases in the activities of Na(+),K(+)-ATPase (to 23% of the control value), Ca(2+)-ATPase (to 16% of control) and Mg(2+)-ATPase (to 22% of control). Washing out the fullerenol lessened the inhibition of the Na(+),K(+)-ATPase (37% of control) and Ca(2+)-ATPase (23.5% of control); however, it did not influence Mg(2+)-ATPase activity. Furthermore, fullerenol could associate with erythrocyte plasma membranes. Our results suggest that fullerenol associates primarily with the surface of the plasma membrane; however, it can also migrate deeper inside the membrane. Moreover, fullerenol influences membrane ATPases so that it may modulate ion transport across membranes.

Can melatonin delay oxidative damage of human erythrocytes during prolonged incubation

PURPOSE Melatonin (MEL) is an effective antioxidant in numerous experimental models, both in vitro and in vivo. However, it should be stressed that there are also papers reporting limited antioxidative activity of MEL or even giving evidence for its pro-oxidative properties. In the present paper we investigated the influence of MEL on the oxidative damage of human erythrocytes during prolonged incubation. MATERIAL/METHODS Human erythrocytes suspended in phosphate-buffered saline (PBS), pH 7.4 were incubated at 37ºC either in absence or presence of melatonin at concentration range 0.02 mM-3 mM for up to 96 hrs. The influence of MEL on erythrocyte damage was assessed on the basis of the intensity of intracellular oxidation processes (the oxidation of HbO₂, GSH, fluorescent label DCFH₂) as well as damage to the plasma membrane (lipid peroxidation, the potassium leakage) and the kinetics of hemolysis. RESULTS The prolonged incubation of erythrocytes induced a progressive destruction of erythrocytes. Melatonin prevented lipid peroxidation and hemolysis whereas the oxidation of HbO₂ and DCFH₂ was enhanced by melatonin at concentrations higher than 0.6 mM. In the case of erythrocytes incubated with 3 mM of MEL, the hemolysis rate constant (0.0498±0.0039 H%•h⁻¹) was 50% lower than that of the control while the HbO₂ oxidation rate constants were about 1.4 and 1.5 times higher for 1.5 and 3 mM of MEL, respectively. Melatonin had no influence on the oxidation of GSH and the potassium leakage. CONCLUSIONS Probably, MEL can stabilize the erythrocyte membrane due to interaction with lipids, thus prolonging the existence of cells. On the contrary, in the presence of MEL the accelerated oxidation of HbO₂ and generally, increased oxidative stress was observed in erythrocytes. Pro- and antioxidative properties of melatonin depend on the type of cells, redox state, as well as experimental conditions.

The Effect of Highly Hydroxylated Fullerenol C60(OH)36 on Human Erythrocyte Membrane Organization

The mechanism of the interaction of highly hydroxylated fullerenol C60(OH)36 with erythrocyte membranes was studied by electron spin resonance spectroscopy (ESR) of stearic acid derivatives labeled with a nitroxyl radical at C-12 or C-16 and with a nitroxyl derivative of maleimide covalently attached to sulfhydryl groups of membrane proteins. A significant increase in membrane fluidity in the hydrophobic region of the lipid bilayer was observed for 12-doxylstearic acid at fullerenol concentrations of 100 mg/L or 150 mg/L, while for 16-doxylstearic acid significant increase in fluidity was only observed at 150 mg/L. Fullerenol at 100 mg/L or 150 mg/L caused conformational changes in membrane proteins, expressed as an increase in the hw/hs parameter, when fullerenol was added before the maleimide spin label (MSL) to the membrane suspension. The increase of the hw/hs parameter may be caused by changes in lipid-protein or protein-protein interactions which increase the mobility of the MSL label and as a result increase the membrane fluidity. Incubation of the membranes with the MSL before the addition of fullerenol blocked the available membrane protein –SH groups and minimized the interaction of fullerenol with them. This confirms that fullerenol interacts with erythrocyte membrane proteins via available protein –SH groups.

Leishmania tarentolae as a host for heterologous expression of functional human ABCB6 transporter.

The need for large amounts of reproducibly produced and isolated protein arises not only in structural studies, but even more so in biochemical ones, and with regard to ABC transporters it is especially pressing when faced with the prospect of enzymatic/transport activity studies, substrate screening etc. Thus, reliable heterologous expression systems/model organisms for large and complex proteins are at a premium. We have verified the applicability of the recently established novel eukaryotic expression system, using Leishmania tarentolae as a host, for human ABC protein overexpression. We succeeded in overexpressing human ABCB6, a protein with controversial subcellular localization and multiple proposed cellular functions. We were able to demonstrate its efficient expression in the expected subcellular locations as well as biochemical activity of the overexpressed protein (ATPase activity and porphyrin-like substrate transport). This activity was absent in cells overexpressing the catalytically inactive variant of ABCB6 (K629M). We demonstrate the possibility of applying a cost-effective expression system to study the activity of membrane transporters from the ABC superfamily.

ABCB1-overexpressing MDCK-II cells are hypersensitive to 3-bromopyruvic acid

AIMS Cancer cells, due to the Warburg effect, are more dependent on glycolysis than normal cells, so glycolytic inhibitor 3-bromopyruvic acid (3-BP) was proposed as a promising candidate for anticancer therapy. Overexpression of multidrug transporters is the main reason of resistance of cancer cells to chemotherapy. As the activity of multidrug transporters imposes an energetic burden on the cells, it can be expected that inhibition of ATP generation may exert a selective cytotoxicity to cells overexpressing multidrug transporters. The aim of this study was to compare the effect of 3-BP on the survival and ATP level in MDCK-II cells and MDCK-II cells overexpressing ABCB1 (Pgp) or ABCG2 (BCRP). MAIN METHODS Cell survival was measured with resazurin and with neutral red. ATP level was assayed with luciferin/luciferase kit. Luteolin transport was measured by an original method described in the paper. KEY FINDINGS 3-BP (10-200μM) induced a decrease of ATP level after 1-h incubation in all cell lines studied, more drastically in ABCB1-overexpressing cells. 50 and 200μM 3-BP significantly decreased cell viability; the effect was more pronounced for ABCB1-overexpressing cells. PSC833, inhibitor of ABCB1, ameliorated the toxic effect of 3-BP on MDCK-II ABCB1 cells and MDCK-II cells. 3-BP inhibited luteolin transport in MDCK-II ABCG2 cells. SIGNIFICANCE These results indicate that 3-BP shows selective toxicity against ABCB1- but not ABCG2-overexpressing cells, apparently due to enhanced ATP depletion but in a manner independent of the transport activity of Pgp, suggesting a novel mechanism of hypersensitivity of ABCB1-overexpressing cells to 3-BP.

Does an anti-oxidant ascorbic acid improve the condition of hippocampal formation slice preparations? – a micro-EEG approach

The objective of this study was to assess whether ascorbic acid (AA), an intracellular anti‐oxidant critical for neuronal protection, when added to artificial cerebrospinal fluid (ACSF), is able to protect hippocampal (HPC) formation slice preparations from ageing. In this research, the micro‐electroencephalographic (EEG) technique was applied. Experiments were performed on 72 HPC formation slices obtained from 12 male Wistar rats. Two series of experiments were conducted: the control experiment, in which ACSF was used as an incubation medium, and the research one, in which ACSF was supplemented with 200 μM AA. The experimental model of carbachol‐induced EEG theta rhythm was applied. The following parameters of theta rhythm after 15, 30 and 45 min of recording were analysed: frequency, power, time duration of theta epochs and time duration of intervals between theta epochs. The results show that AA causes a statistically significant decrease in the power of theta rhythm after 15, 30 and 45 min of recording. The time duration of intervals between theta epochs was almost twice as long in slices incubated in ACSF + AA than in ACSF after 45 min of recording. The data obtained indicate that AA does not improve the condition of HPC slices. On the contrary, it worsens the ability of slice preparations to generate theta oscillations. We hypothesize that our data may result from the Fenton reaction or changes in the conformation of connexins.

Activity of Membrane ATPases in Human Erythrocytes Under the Influence of Highly Hydroxylated Fullerenol

Incubation of erythrocyte membranes with highly hydroxylated fullerenol C60(OH)x, x > 30 led to decreases in Na,K-ATPase, Ca-ATPase and Mg-ATPase activities.