Antitumour and apoptotic effects of a novel Tris-peptide complex obtained after isolation of Raoultella ornithinolytica extracellular metabolites.

AIMS: The characterization of the antitumour activity and chemical identification of the compounds obtained after the isolation of extracellular metabolites of bacteria Raoultella ornithinolytica. METHODS AND RESULTS: The fraction with anticancer activity against the HeLa cell line, T47D and TOV-112D was obtained from the supernatants of R. ornithinolytica culture using ion-exchange chromatography, and separated by Sephadex G-50 medium gel filtration into two subfractions. The obtained compounds were analysed using Fourier Transform-Infrared Spectroscopy, Raman spectroscopy and matrix-assisted laser desorption/ionization MS/MS spectrometry. The antitumour activity of the two subfractions was analysed using 5-bromo-2-deoxy-uridine kit. The subfraction with the highest activity against HeLa cells was identified as Tris-peptide complex. The amino acid sequence of the peptide from the complex was found to be TDAPSFSDIPN and molecular weight was estimated at 1430·6576 Da. Cytotoxic, cytopathic and apoptotic effects in HeLa cells treated with the active complex were observed; however, the cytotoxic effect against normal human skin fibroblasts was minimal. CONCLUSIONS: The Tris-peptide complex from R. ornithinolytica showed selective antitumour activity against the HeLa cell line. SIGNIFICANCE AND IMPACT OF THE STUDY: The Tris-peptide complex due to the high selectivity can be used in biomedicine, and its derivatives may contribute to the development of new anticancer compounds.

Anti-Candida albicans action of the glyco-protein complex purified from metabolites of gut bacterium Raoultella ornithinolytica isolated from earthworms Dendrobaena veneta.

AIMS: The aim of our research was to isolate the compounds from the metabolites of Raoultella ornithinolytica with the activity against Candida albicans and to analyse the action of the compounds on the metabolic activity and morphology of the fungus cells. METHODS AND RESULTS: The effect of active protein fractions on the cell morphology, growth, and metabolic activity of C. albicans was analysed under a light microscope with Nomarski contrast and after staining with calcofluor-white. The LIVE/DEAD Yeast Viability Kit F-7030 FUN 1 was used for determination of C. albicans metabolic activity. The biomolecules obtained after isolation by ion exchange chromatography were further fractionated by Sephadex G-50 medium gel filtration. Then, after molecular sieve, the fractions were analysed by FTIR and SERS Spectroscopy. A subfraction was isolated from the antifungal protein fraction above 100 kDa. The active subfraction identified as the glyco-protein complex caused a decrease in the metabolic activity and morphological changes of C. albicans cells. CONCLUSIONS: The glyco-protein complex obtained from metabolites of bacteria Raoultella ornithinolytica possesses antifungal activity against C. albicans and shows minimal toxicity (1%) against fibroblasts. SIGNIFICANCE AND IMPACT OF THE STUDY: Studies on the glyco-protein complex obtained from earthworm gut bacteria R. ornithinolytica can lead to their application in biological fungicide and pharmaceutical industry.

The effect of quercetin on the expression of heat shock proteins and apoptosis induction in monkey kidney cell line GMK.

The present study was designed to investigate whether quercetin, a very common flavonoid widely distributed in many plants, can induce apoptosis in monkey kidney cells (GMK). Involvement of the expression of heat shock proteins Hsp72, Hsp73, Hsp27 and the significance of cell culture model in this process were also examined. The studies have shown that quercetin alone and in combination with the heat shock can induce apoptosis and necrosis in vitro in the studiedcells, but the percentage of affected cells did not exceed 3.9%. In the same experimental conditions, the expression of Hsp 73, Hsp72 and Hsp27 increased in cells cultured in two-dimensional system and decreased in three-dimensional model. This indicates that strong inhibition of heat shock proteins in GMK cells is not correlated with an adequate increase in the sensitivity of cells to undergo apoptosis. It also shows that the sensitivity on the manifested by Hsp expression and apoptosis induction, depends on the culture model and culture conditions.

The effect of quercetin on apoptosis and necrosis induction in human colon adenocarcinoma cell line LS180.

Quercetin is a very common flavonoid widely distributed in many plants. The flavonoid intake has been linked to the prevention of some human diseases including cancer. Quercetin inhibits heat shock protein expression and in this way triggers apoptosis of tumor cells. The present study was designed to investigate whether quercetin exerts cytotoxic activity against human colon adenocarcinoma cells. The studies have shown that quercetin alone and in combination with the heat shock can induce apoptosis and necrosis in vitro in human colon adenocarcinoma cells (LS 180). Relationships between heat shock proteins and quercetin in this phenomenon are discussed.

The expression of oncogenes in tumour tissues of breast carcinoma patients.

The balance between Bcl-2 and c-Myc and c-Jun seems to be an important determinant of cellular sensitivity to the induction of apoptosis. High expression of Bcl-2 was noticed to be strongly related to low rates of apoptotic cell death. The mean value of the apoptotic index was 45.0% in Bcl-2-negative tumours and 7.5% in Bcl-2-positive tumours. C-Myc and c-Jun accumulation were associated with the absence of Bcl-2 expression and with increased apoptotic activity. The loss of Bd-2 expression was strongly correlated with increased apoptotic cell death. The inverse correlation is between apoptotic and mitotic index. A high mitotic index exists in most patients with a low apoptotic index. Bcl-2, c-Myc and c-Jun does not only take part in cell death, but also in cell division in breast carcinoma cells in which the regulation of cell division and cell death are strictly connected.

Effect of cytotoxic chemotherapy on serum lipid levels in breast cancer patients.

Seventy patients with breast cancer at various clinical stages and 15 patients with fibroadenoma were investigated. After chemotherapy, a significant increase in total triglycerides in malignant breast cancer patients before and after menopause has been observed. The increase in total cholesterol was not significant after chemotherapy. The levels of LDL and HDL cholesterol were significantly decreased in pre- and postmenopausal women treated with chemotherapy.

The effect of aqueous extracts from Inonotus obliquus on the mitotic index and enzyme activities.

The effect of aqueous extract from Inonotus obliquus on the mitotic index and some enzyme activities in human cervical uteri tumour cells HeLa S3 in vitro was evaluated. It was concluded that Inonotus extract inhibited the growth of tumour cells. The fungal extract caused a decrease of the cell protein amount and mitotic index value. Moreover, this extract disturbed metabolism in cells caused decreased activity of LDH, HBDH, MDH, GGT and increasing the activity of catalase.

AgNOR counts and their combination with flow cytometric analyses and clinical parameters as a prognostic indicator in breast carcinoma.

Silver binding nucleolar organizer regions (AgNORs) were counted in tissue sections and were shown to assist in the distinction between benign and malignant breast lesions. The mean AgNOR counts in fibroadenomas were 1.05 +/- 0.85 (n = 18), in lobular carcinoma 3.55 +/- 0.56 (n = 35), and in intraductal carcinoma 4.83 +/- 1.20 (n = 45). AgNOR counting may provide information on breast cancer prognosis supplementary to that obtained with other methods such as flow cytometric analysis. The AgNOR values in carcinomas were also compared with ploidy and S-phase fractions (S) by flow cytometry. More than 75% of the total cancers with counts above 3 dots per nuclear profile contained aneuploid cells, whereas 20% with counts below 3 contained diploid cells (P < 0.05). Similar trends were noticed with regard to S-phase fractions, which were 45% +/- 12.5 and 14% +/- 4.5, respectively, for the two groups (P < 0.05). AgNOR counting, flow cytometry and clinical parameters may provide complementary information on breast cancer prognosis.

Magnesium and iron contents of leukemic lymphocytes in acute leukemias and hemolytic anemia.

In this study, magnesium and iron concentrations were measured in lymphocytes from patients with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and hemolytic anemia (HA) before and after chemotherapy treatment. The results were compared with those of control subjects. Magnesium concentrations were significantly lower in the patient groups, compared with control values. However, no significant differences, except in the HA group, were found among magnesium concentration of the patient groups themselves. Iron level values were at physiological range in all groups. Similarly, no statistically significant differences were found between lymphocyte magnesium concentrations before and after chemotherapy treatment in the patient groups. Fe+3 values were higher in the ALL and HA groups with respect to the group before chemotherapy.

Activities of enzyme transducing extracellular signals--gamma glutamyltransferase and enzymes metabolizing glutathione in acute lymphoblastic and myeloid human leukemias.

The ectoenzyme gamma glutamyltransferase (GGT) a second messenger generating enzyme activity on the cytoplasmic membrane was biochemically analyzed in leukemic cells from patients with acute lymphoblastic and myeloid leukemias. The lower mean activity--0.594 IU/mg protein was noticed in patients with acute lymphoblastic leukemias (ALL), while the higher--0.956 IU/mg protein was found in acute myeloid leukemia patients (AML) in serum and 0.151 IU/mg protein in polymorphonuclear cells. The levels of the activity of glutathione reductase (GR) were increased but the activities of glutathione peroxidase (GSH Px) were significantly decreased in serum of leukemia patients.

DNA index as prognostic factor in breast cancer.

Enzymatically dissociated cells exhibited growth in most cases while mechanically dissociated cells did not grow in short term cultures and were not suitable for cytometric study. Breast cancer cells obtained by enzymatic dissociation were examined for DNA content and S-phase fraction. The most of the primary and metastatic breast cancer cells were aneuploid. The 74% rate of invasive and 62% of primary breast cancers had aneuploid (> G0/G1) DNA content, 22% of metastatic breast cancer cells and 37% of primary tumors cells were DNA diploid or near diploid tumor stemlines. The means of DNA diploid and near diploid tumor cells were higher in primary tumors. DNA index was higher in more aggressive and metastatic tumors. S-phase fractions were higher in cells of metastatic tumors than those in primary tumors. Nuclear DNA content and S-phase fraction can be considered a valuable prognostic indicators in breast cancer.

The effect of low temperature on the enzyme activities and the level of SH groups in benign gastric ulcer and gastric carcinoma.

Homogenates of human benign gastric ulcers and gastric carcinomas were stored at a low temperature (4 degrees C). The activities of L-lactate dehydrogenase (LDH), malic dehydrogenase (MDH), glucose-6-phosphate dehydrogenase (G6PDH), glutathione reductase (GR) and the concentrations of SH-containing compounds showed changes. Increased activities of LDH, MDH, G6PDH and GR occurred in the gastric carcinomas stored at 4 degrees C compared with controls at 37 degrees C. In contrast, the activities of G6PDH and GR decreased in the tissues of benign gastric ulcers. On incubation at 4 degrees C the SH-containing compounds incorporated into protein molecules, SH groups binding with protein and non-protein SH groups decreased in both the benign and malignant tissues.

Enzyme activities and level of SH groups in breast carcinomas.

1. The carcinoma showed higher enzyme activities than the normal mammary tissue. 2. The ratios of glutamate dehydrogenase, glutathione reductase and catalase to lactate dehydrogenase were lower in carcinomas than in normal tissues. Similarly, the ratios of glutamate dehydrogenase, glutathione reductase and catalase to glucose-6-phosphate dehydrogenase were also significantly lower in carcinomas. 3. There were no significant differences in enzyme activities between stages I and II of disease, however in the metastatic tissues, there were significant differences between stages I and II. 4. SH groups were higher in the tissues of cancer patients than in normal tissues. The levels of thiols groups were higher in carcinomas at stage III of disease.

Enzyme activities in human breast tumours.

The activities of six enzymes associated with carbohydrate metabolism were measured both in carcinomas and in normal breast tissues. The following differences were observed. 1. The carcinoma showed higher enzyme activities than the normal mammary tissue. 2. The ratios of glutamate dehydrogenase, hydroxybutyrate dehydrogenase, glutathione reductase and catalase to lactate dehydrogenase were lower in carcinomas than in normal tissues. Similarly, the ratios of glutamate dehydrogenase, hydroxybutyrate dehydrogenase, glutathione reductase and catalase to glucose-6-phosphate dehydrogenase were also significantly lower in carcinomas. 3. There were no significant differences in enzyme activities between I and II stage of the disease and the metastatic tissues, however, there were significant differences between I and III stage. The significance of these findings is discussed in terms of the alterations in the balance between the metabolic pathways.

Effect of the extracts from fungus Inonotus obliquus on catalase level in HeLa and nocardia cells.

Growth medium of Inonotus obliquus exerts antimitotic effect on HeLa cells mostly in M, G1 and G2 phases increasing at the same time catalase activity. This effect was not observed in prokaryotic Nocardia. Significant antimitotic effect of mycelium was not associated with stimulation of catalase activity in HeLa cells.