Цитохаласин B: свойства, применение и влияние на клеточные процессы.

Cytochalasin B, the name of which comes from the Greek cytos (cell) and chalasis (relaxation), is a cell permeable mycotoxin. It was found that substoichiometric concentrations of cytochalasin B (CB) strongly inhibit network formation by actin filaments. Due to this, it is often used in cytological research. It inhibits cytoplasmic division by blocking the formation of contractile microfilaments. It inhibits cell movement and induces nuclear extrusion. Cytochalasin B shortens actin filaments by blocking monomer addition at the fast growing end of polymers. Cytochalasin B inhibits glucose transport and platelet aggregation. It blocks adenosine induced apoptotic body formation without affecting activation of endogenous ADP ribosylation in leukemia HL 60 cells. It is also used in cloning through nuclear transfer. Here enucleated recipient cells are treated with cytochalasin B. Cytochalasin B makes the cytoplasm of the oocytes more fluid and makes it possible to aspirate the nuclear genome of the oocyte within a small vesicle of plasma membrane into a micro needle. Thereby, the oocyte genome is removed from the oocyte, while preventing rupture of the plasma membrane. This alkaloid is isolated from a fungus, Helminthosporium dematioideum.

Cytochalasin B was first described in 1967, when it had been isolated from moulds by Dr W. B. Turner. Smith et al. found that CB causes multinucleation in cells and significantly affects cell motility. The multinucleated cells probably arise from failure of mitotic control, leading to variations in size and shape of interphase nuclei.

In the 1970s, research on the mitosis of polynucleated cells was done. It appeared that these cells were created through progressive nuclear addition instead of nuclear division. The process by which this occurs is called pseudomitosis, which is the synchronous mitosis resulting in the division of just one nucleus. It also affects the appearance of young glands in cells and new gland formation in other cells. Another group found that CB inhibits the ability of HeLa cells to undergo cytokinesis by decomposition of the contractile ring. Research from 1971 showed that CB interferes with the release of iodine derived from thyroglobulin and blocks colloid endocytosis. Moreover, it was found that CB has an inhibitory effect on the uptake of sucrose 3H by chang strain human liver cells and in CB treated cells alterations in the appearance and location of microfilaments were observed. Furthermore, it was found that CB reversibly inhibits melanin granule movement in melanocytes. One year later, research on the influence of cytochalasin B on chloroplasts was done. It was found that the light oriented movement of chloroplasts is reversibly inhibited by cytochalasin B. In 1973 researches found that cytochalasin B is a powerful non competitive inhibitor of glucose transport. One of the major electrophoretic identifiable erythrocyte membrane proteins may be the cytochalasin B binding site of erythrocytes.

In the following years, the knowledge concerning cytochalasin B was broadened. As the more general knowledge had been elucidated, more detailed analysis of e. g. the mechanism of action took place.

Cytochalasins can be isolated from the fungi in which they naturally occur. Originally, they were isolated from Helminthosporium dematioideum. Other producers include Phoma spp., Hormiscium spp. and Curvularia lunata. Additionally, it can be synthesized in the laboratory. There are several approaches to do so. Firstly, it is possible to form the six membered ring of the isoindolone core and the larger macrocyclic ring simultaneously in a late stage intramolecular Diels Alder cyclization. Secondly, it is possible to first form the isoindolone core in an intermolecular Diels Alder reaction and in a second step append the macrocycle in a stepwise fashion.

Cytochalasin B contains several highly polar keto and hydroxyl groups and one peripheric lipophilic benzyl unit.

There are ten possible sites for the in vitro degradation of cytochalasin B. There is not yet any evidence that the same sites are used for degradation in vivo, but evidence has confirmed the in vitro sites. Degradation is initialized by a periodate cleavage of the compound, taking place at carbon 14 and 21. As a result, carbonic acid (A), formaldehyde (B), 5 methylhexane 1,1,6 triol (C) and a large remaining molecule (D) are released. Molecules C and D are then oxidized via Kuhn Roth reaction, leading to the formation of 7 hydroxyheptanal (F), acetic acid (G) and benzoic acid (I). Again, a larger molecule remains (J). F, G and I can undergo Schmidt reaction, if not degraded via acidic degradation by alcohol dehydrogenase (ADH) to methylamine and carbon dioxide (H). 7 hydroxyheptane is oxidized to 3 methylheptanedioic acid (K). Further metabolism leads to the formation of several smaller organic molecules such as amines (M), carbon dioxide (N) and acetic acid (O). The latter is again metabolized by ADH to methylamine and carbon dioxide (Q). Molecule J is cleaved into a number of small compounds such as acetic acid (L), methylamine and carbon dioxide (P), and a series of small methylated compounds. Cytochalasin B is unable to transform 3T3 like tumor cells, but it did increase the frequency of cell transformation by the polyoma virus 8 40 fold. Furthermore, CB can intensify pinocytosis, which is induced by concanavalin A in amoeba proteus. Cytochalasin B can also interact with the auxin indole 3 acetic acid which occurs in wheat coleoptile segments and maize roots. This interaction leads to the inhibition of vesicle transport and secretion of cell wall components and thereby blocks elongation and growth.

In vitro studies showed that a concentration of 30 μM of cytochalasin B significantly reduces the relative viscosity of a 20 μM normal actin filament solution as well as it has decreased in a 20 μMm glutathionyl actin filament solution. In vivo the effective concentration is even lower. It seemed that a 2 μM concentration is sufficient in living cells to accomplish a measurable influence on the actin polymerization. The nucleation phase took 2 4 times as long as in the control groups. On elongation, the effects were minimal; on annealing negligible.

Cytochalasin B is used for testing of the genotoxicity of substances. In order to do so, cytokinesis block micronucleus assay (CBMN assay) with human lymphocytes is applied. This works in vitro. During anaphase of mitosis of meiosis, micronuclei can be detected. These are small nuclei containing one chromosome or part of a chromosome which did not get to one of the cell poles during cell division. The CBMN test is based on the fact that only dividing cells can express micronuclei, which means that only in those cells, chromosome damage can be detected.

Cytochalasin B can decrease the number of motile cells when it is added to Yoshida Sarcoma Cells. It can also decrease the motility of the cells and dose dependently inhibits their growth. Since cytochalasin B unevenly penetrates cells it promotes focal contractions of the broken cortical actin filament network by myosin. This causes superprecipitation which requires active contractions and thus an active energy metabolism. The disorganized cortical contractions disrupt the assembly of pseudopodia which are involved in cell movement.

Nuclear extrusion induced by cytochalasin B begins with the movement of the nucleus to the plasma membrane, followed by bulge formation in the membrane. The nucleus then moves to the outside of the membrane, but stays connected to the cell by a thread like cytoplasmic bridge. If the cells are kept in cytochalasin B containing medium for several hours, the process becomes irreversible. Extrusion could be assisted by the CB induced weakening of the plasma membrane.

It has been shown that cytochalasin B binds covalently to mammalian glucose transporter proteins when irradiated with UV light. It bound tighter to AraE and GalP than their usual substrates. Cytochalasin B has been shown to inhibit GLUT1, 2, 3 and 4. Binding to GLUT1 occurs at the inside as cytochalasin B acts as it acts as a competitive inhibitor of glucose exit. Additional evidence comes from photolabeling studies in which the Trp388 and Trp412 in TM10 and TM11 of the purified protein are labeled upon exposure to labeled cytochalasin B. Since mutating Trp388 and Trp412 does not completely reduce inhibition of GLUT1, it is assumed that other sites are involved in CB binding as well.

For therapeutic purposes, research on cytochalasin B is done. In order to do so, the effects of cytochalasin B on tumor cells by BCG (Bacillus Calmette Guerin) activated macrophages were examined. It showed that cytochalasin B enhances tumor cell lysis and stasis due to activated macrophages at a concentration of 10−7 M. Cytochalasin B does not act on the macrophage itself, but does exert its effect predominantly on the tumor cell. A reason for this could be, that the actin filament formation, which could be important for the destruction of tumor cells by activated macrophages, is inhibited by cytochalasin B.

Cytochalasin B has an effect on thyroid hormone and growth hormone secretion. It does so by inhibiting the conversion of phosphoethanolamine to cytidinediphosphate ethanolamine. It was proposed that the mechanism is associated with alterations of intracellular calcium ions. Cytochalasin B also has effects on bacteria. For example, the growth and differentiation of E. histolytica is inhibited. [56] Furthermore, cytochalasin B has been shown to have an inhibitory effect on tumor cell growth without causing prolonged and/or profound immunosuppressive effects.

In nature, cytochalasin B is involved in fungal virulence, food spoilage and the maintenance of the symbiosis between host and symbiont.