Indole-3-carbinol-induced death in cancer cells involves EGFR downregulation and is exacerbated in a 3D environment

Indole-3-carbinol (I3C) is a promising anticancer dietary compound, which inhibits breast cancer in animal models. The objective of the current study was to characterize I3C-induced cell death in a panel of human breast tumorigenic cells (MCF7, MDA-MB-468, MDA-MB-231 and HBL100) in comparison with normal fibroblasts. Since epithelial cells are protected from cell death by a three-dimensional environment, 3D cell culture (collagen I gel and spheroids) was employed to investigate susceptibility to I3C. Cell viability in the presence of 256 μM I3C, a concentration close to the physiologically achievable range, was in the order fibroblasts = HBL100>MDA-MB-231>MCF7>MDA-MB-468 in monolayer culture. However, 3D culture conditions increased the susceptibility of MCF7 and MDA-MB-468 cancer cells towards I3C. I3C induced cell death in breast cancer MCF7, MDA-MB-468 and MDA-MB–231 cells via the mitochondrial apoptotic pathway. I3C significantly reduced levels of epidermal growth factor receptor (EGFR) in MDA-MB-468 after 6 h and in MDA-MB-231 and HBL100 cells after 30 h. Downregulation of EGFR in MDA-MB468 and MDA-MB-231 cells using an EGFR inhibitor resulted in apoptosis. EGFR modulation using EGF or an EGFR inhibitor markedly influenced viability and response to I3C in MDA-MB-468 cells in 3D conditions. EGFR expression was modulated by 3D conditions. Therefore, I3C-induced EGFR reduction in these cells is likely to be responsible for I3C-induced apoptosis.     

Neutralization of hepatitis B virus surface antigen with immunoglobulin preparations

The in vitro experiments with immunoglobulin and blood plasma containing hepatitis B virus (HBV) markers, revealed that the control of immunoglobulin preparations for the presence of hepatitis B virus surface antigen (HBsAg), mandatory for Russia, was not sufficiently informative. The neutralization of HBsAg with specific antibodies to the level, not determined by the EIA method, reached not less than 24 ng/ ml in 2 hours of incubation and not less than 49 ng/ml in 24 hours of incubation, which, when evaluated in 1 lU of anti-HBs, was 34.6 +/- 0.9 ng and 70.7 +/- 1.8 ng of HBsAg respectively. The process of the formation of immune complexes depended mainly on the time of incubation of experimental samples and on the antibody--antigen proportion in the system. The neutralization of viruses by antibodies had no influence on the capacity of the polymerase chain reaction to detect HBV DNA.     

Specificity of changes in proteasome properties in diethylmaleate-induced apoptosis of K562 cells

The participation of proteasome in the programmed cells death is now extensively investigated. Studies using selective inhibitors of proteasomes have provided a direct evidence of both pro- and anti-apoptotic functions of proteasomes. Such opposite roles of 26S proteasomes in regulation of apoptosis may be defined by the proliferative state of cell. The induction of apoptosis in K562 cells by diethylmaleate was used as a model to investigate changes in the subunit composition, phosphorylation state and enzymatic activities of 26S proteasomes undergoing the programmed cell death. Here we have shown that proteasomes isolated from the cytoplasm of control and diethylmaleate treated K562 cells differ in their subunit patterns, as well as in the phosphorylation state of subunits on threonine and tyrosine residues. It has been shown for the first time that proteolytic activity of 26S proteasomes is decreased, and endoribonuclease activity of 26S proteasomes is affected under diethylmaleate action on K562 cells. Treatment of K562 cells with an inductor of apoptosis--diethylmaleate--leads to modification of a proteasomal subunit (zeta/alpha5) associated with RNase activity of proteasomes. These data suggest the subunit composition and enzymatic activities of 26S proteasomes to be changed in K562 cells undergoing apoptosis, and that specific subtypes of 26S proteasomes participate in execution of programmed death of these cells.     

Use of stable analogs of myosin ATPase intermediates for kinetic studies of the "weak" binding of myosin heads to F-actin.

It is known that ternary complexes of myosin subfragment 1 (S1) with ADP and the Pi analogs beryllium fluoride (BeFx) and aluminum fluoride (AlF4-) are stable analogs of the myosin ATPase intermediates M* x ATP and M** x ADP x Pi, respectively. Using kinetic approaches, we compared the rate of formation of the complexes S1 x ADP x BeFx and S1 x ADP x AlF4- in the absence and in the presence of F-actin, as well as of the interaction of these complexes with F-actin. We show that in the absence of F-actin the formation of S1 x ADP x BeFx occurs much faster (3-4 min) than that of S1 x ADP x AlF4- (hours). The formation of these complexes in the presence of F-actin led to dissociation of S1 from F-actin, this process being monitored by a decrease in light scattering. The light scattering decrease of the acto-S1 complex occurred much faster after addition of BeFx (during 1 min) than after addition of AlF4- (more than 20 min). In both cases the light scattering of the acto-S1 complex decreased by 40-50%, but it remained much higher than that of F-actin measured in the absence of S1. The interaction of the S1 x ADP x BeFx and S1 x ADP x AlF4- complexes with F-actin was studied by the stopped-flow technique with high time resolution (no more than 0.6 sec after mixing of S1 with F-actin). We found that the binding of S1 x ADP x BeFx or S1 x ADP x AlF4- to F-actin is accompanied by a fast increase in light scattering, but it does not affect the fluorescence of a pyrene label specifically attached to F-actin. We conclude from these data that within this time range a "weak" binding of the S1 x ADP x BeFx and S1 x ADP x AlF4- complexes to F-actin occurs without the subsequent transition of the "weak" binding state to the "strong" binding state. Comparison of the light scattering kinetic curves shows that S1 x ADP x AlF4- binds to F-actin faster than S1 x ADP x BeFx does: the second-order rate constants for the "weak" binding to F-actin are (62.8 +/- 1.8) x 10(6) M-1 x sec-1 in the case of S1 x ADP x AlF4- and (22.6 +/- 0.4) x 10(6) M-1 x sec-1 in the case of S1 x ADP x BeFx. We conclude that the stable ternary complexes S1 x ADP x BeFx and S1 x ADP x AlF4- can be successfully used for kinetic studies of the "weak" binding of the myosin heads to F-actin.     

Therapeutic effect of a single peritumoural dose of IL-2 on transplanted murine breast cancer

Interleukin-2 therapy is not clearly effective against breast cancer both in mouse models and in human patients. However, the study of IL-2 therapy of breast cancer remains important, as 3,700 women died from this malignancy in the Netherlands in 2000. Previously we have shown the therapeutical efficacy of a single peritumoural IL-2 application in different experimental models and in veterinary patients. Here we apply this mode of IL-2 therapy to advanced mouse mammary carcinoma models, i.e., severe metastasised tumours in A/Sn mice and non-metastasised carcinomas in BALB/c mice. Mice with advanced transplanted mammary carcinomas were given a single peritumoural treatment with 2.5 x 10(6) IU IL-2 at days 10-14 after i.p. or s.c. inoculation of 10(6) carcinoma cells. Within each experiment it was always possible to distinguish relatively slowly and fast growing tumours which allows the therapeutical effect of IL-2 in tumours with different growth rates to be studied. A new approach to analyse results enabled us to show that survival of mice with transplanted, advanced metastasised breast cancer can be significantly improved after a single local treatment with IL-2. Advanced relatively fast i.p and s.c. growing mammary carcinomas seem to be more sensitive to a single IL-2 treatment than relatively slowly growing tumours. IL-2 was most effective against non-metastasised mouse breast cancer.     

Modelling of myocardial hypertrophy in vitro for solving problems of medicinal correction

The work has been done on primary heart culture from neonatal rat ventricle. Cardiomyocyte hypertrophy was modelled using noradrenaline (NA), angiotensin II (AII) and fetal serum, respectively. Cell hypertrophy of primary heart cultures was assessed by measuring the surface area, the scope of protein synthesis estimated by 3H-leucine autoradiography and the contents of nucleic acids in gallocyanin-chromalum stained cardiomyocytes. The structure of myofibrillar apparatus was studied by rhodamine-conjugated phalloidin and indirect immunofluorescence of muscle alpha-actinin. Treatment with 10(-6) M NA increased 3H-leucine incorporation in 9-day old heart culture by 42% without changing cell size. AII in a dose 1 microM stimulated protein synthesis activity by 1.3 fold and the surface area by 1.7 fold, both in 2- and 9-day old primary heart cultures. The maximum stimulation of cell hypertrophy was provided by the medium supplemented with fetal serum. RNA contents in the cytoplasm of cardiomyocytes increased by 7.8 fold and the myocardial cell size by 2.9 fold in serum-supplemented culture by 9 days of cultivation. In the medium with fetal serum, amounts of cardiomyocytes with tetraploid nuclei reached 33%, against 14% in control. Coculturing of myocardiocytes and fibroblasts rendered effects of fetal serum on the growth of myocardiocytes. Cultivation in the presence of 1 microM enalapril, an ACE inhibitor, suppressed the development of cardiac muscle cells hypertrophy. The effect of enalapril depended on the degree of cellular hypertrophy. Addition of 10 microM amiloride to the medium lowered the protein synthesis by 29% independently on the initial cellular hypertrophy.     

New data on the Cenomanian Flora of the Ugol’naya bay (Northeastern Russia)

The taxonomic composition of the Middle Ginter Flora of the Ugol’naya Bay area (northeastern Russia) is supplemented with new data based on newly determined and revised materials. The uniqueness of the flora lies in the fact that its age (Middle Cenomanian) is dated to a zone by marine fauna. The floristic assemblage contains 29 species of fossil plants and is dominated by angiosperms and conifers. The refined taxonomic list shows even greater than was earlier supposed similarity of the flora to the Grebenka assemblage from the Krivorechenskaya Formation at the left bank of the Anadyr River, which is the type assemblage of the Grebenka phase of the flora development in northeastern Russia. A new combination is proposed: Ettingshausenia louravetlanica (Herman et Shczepetov) Herman et Moiseeva, comb. nov.     

The density of myocardial and pericardial rat mast cells in isoproterenol-induced heart failure

The multifunctional mast cells (MC), located in the myocardium, actively react to the pathology of a cardiovascular system. We investigated MC density in the pericardium and myocardium of rats in intact and experimental heart failure (HF) 24 h and 14, 28, and 60 days after two (at 24 h intervals) injections of Isoproterenol (IP) inducing necrosises in the myocardium. An expressiveness of HF was estimated with the help of ultrasonic scanning of the heart after 28 and 60 days after two IP injections. MC of different degrees of maturity were identified cytochemically on paraffin sections stained with Alcian blue-Safranine. The MC density in the myocardium of intact and experimental rats was relatively low, ranging from 4 to 6 cells/mm², thus the ratio of cells of a different degrees of maturity during HF development was reliably unchanging. In the fibrous layer of pericardium, the MC density was higher than in the myocardium and, for the intact rats, it amounted 48.6 ± 13.0 cells/mm². 24 h to 14 days after IP injections, the MC density in pericardium in contrast to intact sample increased on the average in 1.5 times (P < 0.05) at the expense of increase of density of more differentiated cells stained with Safranine, without density change of less differentiated cells stained with Alcian blue. After 28 days, the MC density in pericardium was 2 times higher than in the intact sample (P < 0.01) and, at the same time, the density of cells of a different maturity degree (P < 0.05) was considerably increased. For 60 days, the MC density and the balance of Alcian-and Safranine-positive cells did not differ reliably from the intact sample. The dynamic of behavior of the MC population of pericardium was corresponding to HF injury on functional parameters. The data obtained indicate the active reaction of MC pericardium on a dysfunction of the myocardium; it stimulates the maturation of resident MCs and the reinforcement of the population at the expense of the migration of cells from the outside, which allows us to propose an intensification of the MC secretory function.