Biomedical subjects

E Lyubimov

Publications and source records attributed to E Lyubimov.

6 recordsLinked to original sources

PubMed2006

Thrombin-triggered platelet apoptosis.

BACKGROUND: Thrombin is primarily known as a coagulation factor and as an inducer of platelet activation and aggregation. It has been reported that thrombin modulates apoptosis of nucleated cells. OBJECTIVES: The current study investigated whether thrombin can affect apoptosis in anucleated human platelets. METHODS: Using flow cytometry, we studied platelet apoptosis at the single-cell level, analyzing markers of mitochondrial and cytoplasmic apoptosis. Western blotting was also employed, in addition to flow cytometry, for determining the expression of Bcl-2 family proteins. RESULTS: We found that human alpha-thrombin induced four key manifestations of apoptosis in human platelets: (i) mitochondrial inner transmembrane potential (DeltaPsi m) depolarization; (ii) strong expression of pro-apoptotic Bax and Bak proteins but only weak expression of anti-apoptotic Bcl-2 protein; (iii) caspase-3 activation; and (iv) phosphatidylserine (PS) exposure. CONCLUSIONS: This study demonstrates that, aside from its 'classical' function as an inducer of platelet activation, thrombin can trigger platelet apoptosis, where it acts as a death ligand. These data indicate that thrombin triggers platelet apoptosis by impacting on several intracellular apoptotic targets, including shifting the balance between Bcl-2 regulatory proteins in a pro-apoptotic direction, depolarizing the inner mitochondrial membrane, activating the executioner caspase-3, and stimulating aberrant exposure of PS on the platelet surface.

Apoptosis↗

PubMed2000

Synergistic effect of prochlorperazine and dipyridamole on the cellular retention and cytotoxicity of doxorubicin.

Incubation of drug-resistant human tumor cells with a combination of prochlorperazine and dipyridamole has additive/synergistic effect on the cellular retention and cytotoxicity of doxorubicin. In patients administered a fixed dose of doxorubicin and prochlorperazine with escalating doses of dipyridamole, mean plasma levels of dipyridamole and prochlorperazine achieved were as high as 3.01 +/- 0.41 microm and 0.94 +/- 0.09 microm, respectively. Plasma samples from patients were analyzed in an in vitro assay to monitor the effect on the cellular retention of tritium-labeled daunorubicin in MDR1-transfected P388 cells. In 22 of 49 of the plasma samples analyzed, the daunorubicin in efflux blocking activity was one-half or greater than that of cells incubated with 12.5 microM verapamil, a well-known efflux blocker. These observations suggest that a combination of prochlorperazine and dipyridamole may enhance cellular doxorubicin retention by blocking efflux while reducing normal tissue toxicity and unwanted side effects in vivo.

Animals↗

PubMed1996

Kinetic parameters for reversal of the multidrug pump as measured for drug accumulation and cell killing.

We determined the kinetic parameters that describe the effect of 20 different modulators of the multidrug resistance pump on the reversal of cytotoxin accumulation in a resistant strain of P388 leukemia cells (P388/ADR), and on the reversal of cell killing for these cells. When measured by a direct comparison of the amplitude of the pertinent protocol (accumulation or cell killing), the Ki for reversal of accumulation was generally some four or five times larger than that for reduction of cytotoxicity. We showed that this was only an apparent discrepancy, since a full theoretical analysis of the two protocols allowed the intrinsic Ki to be obtained for the two procedures and these computed Ki values were then almost identical. We found that for six of the modulators studied (namely, cyclosporin A, quinidine, dipyridamole, propafenone, mefloquine, tamoxifen) the extent of pump reversal should be better than 90% at tolerated plasma levels culled from the literature.

Antineoplastic Agents↗

PubMed1996

Effect of modulators of the multidrug resistance pump on the distribution of vinblastine in tissues of the mouse.

Vinblastine at doses ranging from 0.2 to 6 mg/kg body weight was administered i.p. to mice in the absence or presence of the drugs PSC 833, cyclosporin A, mefloquine, quinidine and dipyridamole, all compounds that modulate the multidrug resistance pump and thus increase the accumulation of this cytotoxin in drug-resistant cells in cell culture. In the absence of modulators, vinblastine accumulated in tissues to different extents--lowest in brain, highest in pancreas and intestine. The extent of accumulation was directly proportional to the vinblastine dose in the range 0.2-6 mg/kg body weight. Both at high and low vinblastine doses, all the modulators except quinidine increased the ability of liver, kidney, intestine and lung to accumulate vinblastine by up to 5-fold, and with the further exception of mefloquine, also increased vinblastine levels in pancreas. Only dipyridamole had a marked effect also in brain. Cyclosporin A provided effective increases in the tissue distribution of vinblastine at plasma concentrations similar to those needed to block the multidrug pump in the case of cells in cell culture. For mefloquine, plasma concentrations three or four times higher were needed in vivo than were found to be effective in cell culture. The mouse system provides a quick and reliable in vivo method to assay modulators before they are tested in the clinic.

Animals↗

PubMed1996

Reversal of P-glycoprotein is greatly reduced by the presence of plasma but can be monitored by an ex vivo clinical assay.

The effects of nine reversers of P-glycoprotein on the uptake of daunomycin into MDR1-transfected P388 cells were quantitatively determined in undiluted human or mouse plasma and compared with their effects when measurements are made in a conventional cell culture medium (RPMI 1640) containing only 10% serum. Plasma diminished or greatly diminished the effectiveness of the reversers, reductions of up to 20-fold being found for reversers (cyclosporin A, prochlorperazine and amiodarone) that have been used in clinical trials, although quinidine was almost as effective in plasma as in cell culture medium containing 10% fetal calf serum. Human or bovine serum albumin could mimic the effect of whole plasma. When measurements of the effectiveness of the reverser cyclosporin A were made in an ex vivo assay, using these P388 cells, complete accord was found between such ex vivo determinations and cyclosporin A's effectiveness in vivo, as monitored by its ability to increase the accumulation of vinblastine in mouse kidney tissue. The ex vivo assay was shown to be suitable to monitor the effectivity of reversers present in plasma taken from patients receiving quinidine and cyclosporine A in routine clinical treatment.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Saturation reversal of the multidrug pump using many reversers in low-dose combinations.

Multidrug resistance in cancer cells, in cell culture and in the clinic, is often associated with a membrane protein (the multidrug resistance pump or P-glycoprotein) that pumps out anti-cancer drugs as fast as they enter the cell. This pump is blocked by a range of well-known pharmaceuticals that reverse drug resistance. We have investigated whether effective reversal of drug resistance could be achieved by using many reversers together, each at a low dose relative to its maximal tolerated plasma level. We measured in cell culture, using resistant P388 cells in suspension, the extent of reversal of the accumulation of two labeled cytotoxins (vinblastine and daunomycin). We fitted the data to a modified Michaelis-Menten equation and extracted the half-inhibition constants for 18 reversers acting on the pump. We measured also the reversal of resistance in a cell growth assay using incorporation of labeled thymidine. We showed that these drugs in groups of up to 18 together, each drug being at a low dose, in many cases well-tolerated in humans, had additive effects so that the combination was as effective as any of the drugs present singly. This was the case both for reversal of cell accumulation and for the effects of cytotoxins on cell growth. Our data show that a low-dose multidrug approach to saturation reversal of the multidrug pump is feasible in cell culture and provide the initial experimental basis for the development of an effective regime of such combination reversal therapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗