Effects of antineoplastic agents on growth,morphology and metabolism of Torulopsis glabrata

The effect of treatment by a number of antineoplastic agents on the growth, ultrastructure and macromolecular synthesis of T. glabrata was studied. Many differences were noted in the response of this yeast to these agents. Thiotepa and methotrexate inhibited the growth of T. glabrata, while it was resistant to endoxan-asta and vincristine sulphate.A variation of morphological response of T. glabrata was also observed. Methotrexate enhanced filamentation while thiotepa influenced the surface structures of the cells, resulting in loss of cytoplasmic materials and cell collapse. The other two drugs had little or no effect on the morphology of the yeast tested.The incorporation of precursors for macromolecular synthesis of T. glabrata in the presence of thiotepa and methotrexate was restricted. Thiotepa affected the uptake of precursors of RNA, DNA and protein limiting them to between 62 and 66% of the control values. In contrast, methotrexate limited the uptake of macromolecular precursors to a lesser extent. The possible mechanism of action of antineoplastic agents against yeast and the clinical implications of these findings are discussed.     

Effect of antineoplastic agents and X-irradiation on the adherence of Candida spp. to human buccal epithelial cells in vitro

The role of chemotherapy, X-irradiation and a combination of both on the phenomenon of adherence of yeast to buccal epithelial cells (BEC) was investigated in vitro. Growth of three Candida spp. in the presence of eight of eleven antineoplastic agents led to reduction of adherence of the isolates tested (reduction between 30% and 61% of the control value), and this effect was observed whether exponential or stationary phase Candida cells were used. Exposure of C. albicans to various doses of radiation also led to a reduction in adherence of this yeast to BEC between 31% and 53% of the control value. This reduction was shown to be dose related. Similar results were obtained when BEC were exposed to radiation, and the effect of radiation treatment was accentuated when both yeast and BEC were irradiated simultaneously. Furthermore, treating C albicans with a combination of chemotherapy and radiation led to the greatest reduction in adherence of yeast to BEC compared to when the yeast was treated with either chemotherapy or radiation alone (reduction between 63% to 74% as compared with control). The possible mechanism/s involved in reduction of adherence of yeast to BEC are discussed.     

Anticancer drugs as inducers of thermotolerance in yeast

Yeast cell viability was evaluated microscopically following exposure to heat shock for 30 min at 53°C. The cells were previously grown in the presence of potential stressors (anticancer drugs;e.g., 5-fluorouracil, methotrexate, cisplatin, bleomycin, mitomycin-C and camptothecin-11). The induction of thermotolerance was documented by significantly increased viability after heat shock. This effect, which was reversed by cycloheximide, was comparable to that observed following exposure to a mild heat stress. These data demonstrate that pretreatment with sub-toxic concentrations of some of the clinically used antineoplastic agents conferres thermotolerance to yeast, possibly through the synthesis of protein components.     

Direct interaction between the catalytic subunit of Protein Phosphatase 1 and pRb

Background

Among the epigenetic alterations occurring in cancer, DNA hypermethylation and histone hypoacetylation are the focus of intense research because their pharmacological inhibition has shown to produce antineoplastic activity in a variety of experimental models. The objective of this study was to evaluate the combined antineoplastic effect of the DNA methylation inhibitor hydralazine and the histone deacetylase inhibitor valproic acid in a panel of cancer cell lines.

Results

Hydralazine showed no growth inhibitory effect on cervical, colon, breast, sarcoma, glioma, and head & neck cancer cell lines when used alone. On the contrary, valproic acid showed a strong growth inhibitory effect that is potentiated by hydralazine in some cell lines. Individually, hydralazine and valproic acid displayed distinctive effects upon global gene over-expression but the number of genes over-expressed increased when cells were treated with the combination. Treatment of HeLa cells with hydralazine and valproic acid lead to an increase in the cytotoxicity of gemcitabine, cisplatin and adriamycin. A higher antitumor effect of adriamycin was observed in mice xenografted with human fibrosarcoma cells when the animals were co-treated with hydralazine and valproic acid.

Conclusion

Hydralazine and valproic acid, two widely used drugs for cardiovascular and neurological conditions respectively have promising antineoplastic effects when used concurrently and may increase the antitumor efficacy of current cytotoxic agents.     

Influence of cultivation procedure for <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> used as pitching agent in industrial spent sulphite liquor fermentations

The cell viability and fermentation performance often deteriorate in fermentations of spent sulphite liquor (SSL). This investigation therefore addresses the question of how different cultivation conditions for yeast cells influence their ability to survive and boost the ethanol production capacity in an SSL-based fermentation process. The strains used as pitching agents were an industrially harvested Saccharomyces cerevisiae and commercial dry baker’s yeast. This study therefore suggests that exposure to SSL in combination with nutrients, prior to the fermentation step, is crucial for the performance of the yeast. Supplying 0.5 g/l fresh yeast cultivated under appropriate cultivation conditions may increase ethanol concentration more than 200%.     

Synthetic Studies on 2′-Substituted-4′-thiocytidine Derivatives as Antineoplastic Agents

Abstract

As potential antineoplastic agents, we have synthesized 4′-thioFAC and 4′-thiocytarazid by developing an alternative synthetic method. 4′-ThioFAC showed potent antineoplastic activities in vivo as well as in vitro.     

Inhibition of benzo(a)pyrene-induced carcinogenesis by vitamin C alone and by vitamin C/vitamin E and selenium/glutathione

The reduction on peroxidation caused by benzopyrenes by some naturally occurring antineoplastic agents was studied in this experimental work. Inhibition/reduction of experimental carcinogenesis induced by benzo(a)pyrene by vitamin C alone and by vitamin C/vitamin E and selenium/glutathione was attempted in 224 female Wistar rats divided in four groups. Injected with 10.08 mL benzo(a)pyrene, the animals were treated with some naturally occurring substances like vitamin C alone and a combination of anticarcinogens. By calculating the carcinogenic potency of benzo(a)pyrene and the anticarcinogenic potency of substances used as well as histological examination of developed tumors and survival time of treated animals, it was found that vitamin C exerts a significant anticarcinogenic effect of 8.3 units and that the combination of the two anticarcinogens used produced a significant prolongation of the animals survival time with anticarcinogenic potency of 22.1 and 22.2 units, respectively. This is considered a potent anticarcinogenic effect. The question of an additional supportive administration of such agents complementary to the conventional cancer chemotherapy in humans is raised. Of course, further studies are needed.     

Effect of calcium channel blockers on the action of various antitumour agents in the yeast Saccharomyces cerevisiae

The cytotoxic effects of a 22 h treatment with four antineoplastic agents in the yeast Saccharomyces cerevisiae ATCC 2366 were investigated. Two agents, doxorubicin and 5-fluorouracil (5-FU), were effective in decreasing the colony-forming ability of yeast cells. Following examination under the light microscope, the effect of doxorubicin appeared to be, at least partially, due to killing of yeast cells whereas the effect of 5-FU was rather due to changes in cell structure leading to abnormal bud formation. For amsacrine (AMSA) and melphalan, cytotoxicity was totally absent. In the presence of diltiazem the above described effects were not significantly changed. When verapamil was added in the culture medium the cytotoxic activity of doxorubicin and 5-FU did not change. However, following treatment with AMSA in combination with verapamil, cell survival was significantly decreased whereas the presence of verapamil increased the yeast survival which was observed after melphalan treatment.     

No effect of 50 Hz 2.45 mT magnetic field on the potency of cisplatin, mitomycin C, and methotrexate in S. cerevisiae

Drug resistance is an obstacle for chemotherapy success. Because of this, this work aims to improve the cell killing effect of antineoplastic drugs by magnetic field (MF) co-exposure. S. cerevisiae cells were exposed to 2.45 mT, sinusoidal 50 Hz MF, during 48 h, and the drugs cisplatin (cisPt), mitomycin C (MMC), or methotrexate (MTX); 100 and 1,000 microg/ml. Survival was assayed by the drop test. The results showed that MF exposures do not induce alterations in the potency of cisPt, MMC, and MTX on these cells in relation to untreated controls. In addition, a strong correlation between temperature and potency of cisPt was found, which contribute to the establishment of the importance of an exhaustive control of temperature in experiments carried out with temperature sensitive antineoplastic agents in co-exposure with MF; avoiding differences between MF-exposed samples and unexposed controls and contributing to the performance of experiments under well-defined and controlled conditions.     

The Synergistic Effects among β-,3-Glucanases from Oerskovia sp. CK on Lysis of Viable Yeast Cells

Oerskovia sp. CK produced three types of β-1,3-glucanases designated as F-L, F-0 and F-2. F-L showed high lytic activity to viable yeast cells and weak activity to yeast glucan. F-0 and F-2 had little or no lytic activity and strong β-1,3-gIucanase activity.

F-0 or F-2 showed high lytic activities to yeast cells pretreated with small amounts of F-L which did not lysed the cells. Lytic activity of F-0 or F-2 also increased when cells were treated with alkaline pH or with both reducing agents and pH.

From these results, it is supposed that the ineffectiveness of F-0 or F-2 on the lysis of yeast cells might be attributed to a spatial inaccessibility of enzymes to the yeast glucan layer. However, the treatment of F-L, alkaline pH and reducing agents would bring about a modification of cells to give F-0 or F-2 access to the wall glucan and consequently the lysis of cells would occur.     

Evaluation of different formulations of potential biocontrol yeast isolates efficacy on apple blue mold at storage condition

Post-harvest pathogens cause major losses in apple production. Biological control by using epiphytic yeasts against Penicillium expansum has been considered as an alternative method for controlling the post-harvest decays. The yeast isolates Rhodotorula mucilaginosa, Pichia guilliermondii, which showed high biocontrol efficacy against P. expansum, were selected for formulation tests. Formulation is an important step in developing a biocontrol product. The successful delivery of biocontrol agents, shelf life, stability and effectiveness in commercial conditions depend on the formulation. In the formulation, the carrier is the primary material used to allow a bioproduct to be dispersed effectively. Yeast isolates were grown in a cane molasses-based medium. Harvested yeast cells were combined with inorganic (talk, kaolin) and organic (Rice bran, wheat bran) carriers. Viability of the yeast cells in formulations stored at 4°C and 24°C was determined each month during 6 months storage. After 6 months storage to evaluate efficacy of formulations, all formulations were tested on apple to control blue mold in storage condition. High stability of antagonistic yeasts was achieved by using organic and inorganic carriers. Rice bran and wheat bran stimulated the germination of the yeasts cells during storage period. Both of the yeasts had significantly highest viable yeast cell content over 6 months in formulation containing wheat bran as a carrier. P.guillermondii in all formulations had significantly higher shelf life and was effective than R. mucilaginosa.     

‘Yeast Mail’: A Novel Saccharomyces Application (NSA) to Encrypt Messages

The universal genetic code is used by all life forms to encode biological information. It can also be used to encrypt semantic messages and convey them within organisms without anyone but the sender and recipient knowing, i.e., as a means of steganography. Several theoretical, but comparatively few experimental, approaches have been dedicated to this subject, so far. Here, we describe an experimental system to stably integrate encrypted messages within the yeast genome using a polymerase chain reaction (PCR)‐based, one‐step homologous recombination system. Thus, DNA sequences encoding alphabetical and/or numerical information will be inherited by yeast propagation and can be sent in the form of dried yeast. Moreover, due to the availability of triple shuttle vectors, Saccharomyces cerevisiae can also be used as an intermediate construction device for transfer of information to either Drosophila or mammalian cells as steganographic containers. Besides its classical use in alcoholic fermentation and its modern use for heterologous gene expression, we here show that baker's yeast can thus be employed in a novel Saccharomyces application (NSA) as a simple steganographic container to hide and convey messages.     

Flavonoids from <Emphasis Type="Italic">Heliotropium subulatum</Emphasis> exudate and their evaluation for antioxidant,antineoplastic and cytotoxic activities II

The flavonoids are the largest group of phenolic compounds isolated from a wide range of higher plants. These compounds work as antimicrobials, anti-insect agents and protect plants from other types of biotic and abiotic stresses. Various researchers have suggested that flavonoids possessed antioxidant, antineoplastic and cytotoxic activities. The main objective of this study was to test dichloromethane fraction of resinous exudate of Heliotropium subulatum for their antioxidant, antineoplastic and cytotoxic activities, as well as to search new antioxidant and antineoplastic agents for pharmaceutical formulations. Five flavonoids were isolated from resinous exudate of this plant species and screened for their in vitro and in vivo antioxidant models (DPPH radical scavenging, reducing power, superoxide anion scavenging, metal chelating scavenging systems, catalase and lipid peroxidation), antineoplastic (Sarcoma 180), and cytotoxic (Chinese hamster V79 cells) activities. Tricetin demonstrated maximum antioxidant activity against both in vitro and in vivo experimental systems while galangin exhibited maximum inhibition (78.35%) at a dose of 10 µg/kg/day against Sarcoma 180. Similarly, it was found that galangin also showed highest activity (21.1 ± 0.15%) at a concentration of 70 µg/ml to Chinese hamster V79 cells. The observed results suggest that tricetin has a potential to scavenge free radicals in both in vitro and in vivo models while the galangin could be considered as antitumor and cytotoxic agent.     

Overexpression of bacterioferritin comigratory protein (Bcp) enhance viability and reduced glutathione level in the fission yeast under stress

The structural gene encoding bacterioferritin comigratory protein (Bcp) was amplified using PCR from the genomic DNA of Schizosaccharomyces pombe, and transferred into the shuttle vector pRS316 to generate the recombinant plasmid pBCPlO. The bcp ⁺ mRNA level in the pBCPlO-containing yeast cells was significantly higher than that in the control yeast cells, indicating that the cloned gene is functioning. The S. pombe cells harboring the plasmid pBCPIO exhibited higher survival on the solid minimal media with hydrogen peroxide, tert-BOOH or cadmium than the control yeast cells. They also exhibited enhanced cellular viability in the liquid media containing the stressful agents. The increased viabilities of the fission yeast cells harboring the plasmid pBCP10 were also obtained with 0.4% glucose or 0.4% sucrose as a sole carbon source, and nitrogen starvation, compared with those of the control yeast cells. The total glutathione (GSH) content and total GSH/GSSG ratio were significantly higher in the yeast cells harboring the plasmid pBCP10 than in the control yeast cells. In brief, the S. pombe Bcp plays a protective role in the defensive response to oxidative stress possibly via up-regulation of total and reduced glutathione levels.     

Selected dehydrogenases in Yarrowia lipolytica JMY 861: their role in the synthesis of flavor compounds

The presence of selected dehydrogenases, including alcohol dehydrogenase (ADH-YL) and aldehyde dehydrogenase (ALDH-YL), in Yarrowia lipolytica JMY 861, and their potential role in flavor synthesis were investigated. The experimental findings showed that using reduced form of nicotinamide adenine dinucleotide (NADH) as cofactor, the ADH-YL activity in vitro was 6-fold higher than that with reduced form of nicotinamide adenine dinucleotide phosphate (NADPH); however, under the experimental conditions used in this study, an ALDH-YL activity was not detected. The in situ hexanal reduction reaction was found to be instantaneous; however, when the yeast cells suspension was diluted 150 times, the initial relative hexanal concentration was increased by 84.1%. The chromatographic analyses indicated the conversion, in situ, of linoleic acid hydroperoxides (HPODs) into volatile C₆-compounds after 60 min of HPODs addition to the yeast cells suspension.     

Vital imaging of H9c2 myoblasts exposed to <Emphasis Type="Italic">tert</Emphasis>-butylhydroperoxide – characterization of morphological features of cell death

Background  

When exposed to oxidative conditions, cells suffer not only biochemical alterations, but also morphologic changes. Oxidative stress is a condition induced by some pro-oxidant compounds, such as by tert-butylhydroperoxide (tBHP) and can also be induced in vivo by ischemia/reperfusion conditions, which is very common in cardiac tissue. The cell line H9c2 has been used as an in vitro cellular model for both skeletal and cardiac muscle. Understanding how these cells respond to oxidative agents may furnish novel insights into how cardiac and skeletal tissues respond to oxidative stress conditions. The objective of this work was to characterize, through vital imaging, morphological alterations and the appearance of apoptotic hallmarks, with a special focus on mitochondrial changes, upon exposure of H9c2 cells to tBHP.     

Comparison of various ways of extraction of nucleic acids and of preparation of yeast extract from saccharomyces cerevisiae and candida utilis

Six different variations of the extraction procedure applied to yeast cells of Saccharomyces cerevisiae and Candida utilis to optimize the production of yeast extract and isolation of nucleic acids were compared. The autolysis of C. utilis at 50 to 52°C without adding chemical agents was found to be the best for the production of yeast extract. The most suitable procedures used for the extraction of nucleic acids were those which were carried out from C. utilis at pH 7.5 (92°C) and the other with 0.4 M NH₄OH (40°C). Both these modifications yielded the highest amounts of polymer nucleic acids. Applying all procedures compared to S. cerevisiae an increased content of sterols (including Δ^5.7-sterols, predominantly ergosterol) was detected.     

Unravelling the role of SNM1 in the DNA repair system of Trypanosoma brucei

All living cells are subject to agents that promote DNA damage. A particularly lethal lesion are interstrand cross‐links (ICL), a property exploited by several anti‐cancer chemotherapies. In yeast and humans, an enzyme that plays a key role in repairing such damage are the PSO2/SNM1 nucleases. Here, we report that Trypanosoma brucei, the causative agent of African trypanosomiasis, possesses a bona fide member of this family (called TbSNM1) with expression of the parasite enzyme able to suppress the sensitivity yeast pso2Δ mutants display towards mechlorethamine, an ICL‐inducing compound. By disrupting the Tbsnm1 gene, we demonstrate that TbSNM1 activity is non‐essential to the medically relevant T. brucei life cycle stage. However, trypanosomes lacking this enzyme are more susceptible to bi‐ and tri‐functional DNA alkylating agents with this phenotype readily complemented by ectopic expression of Tbsnm1. Genetically modified variants of the null mutant line were subsequently used to establish the anti‐parasitic mechanism of action of nitrobenzylphosphoramide mustard and aziridinyl nitrobenzamide prodrugs, compounds previously shown to possess potent trypanocidal properties while exhibiting limited toxicity to mammalian cells. This established that these agents, following activation by a parasite specific type I nitroreductase, produce metabolites that promote formation of ICLs leading to inhibition of trypanosomal growth.     

The metal-coordinated Casiopeína IIIEa induces the petite-like phenotype in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The Casiopeínas^® are mixed chelate copper (II) complexes and promising antineoplastics agents against cancer cells and tumors in vitro and in vivo. However, the action mode of these compounds is poorly characterized. In this work the effect of the antineoplastic Casiopeína IIIEa on the metabolism and ultrastructure of the yeast Saccharomyces cerevisiae was investigated. Exposure of cells growing in rich or in low-iron medium to 5 μM of the compound decreased duplication time and reduced oxygen consumption. Those cells formed smaller colonies when growing in a non-fermentable carbon source and low-iron medium, and under the light microscope, multiple folds were observed along the plasma membrane accompanied with a reduction in the diameter of the yeast. These observations were confirmed under the electron microscope, which also revealed a slight reduction of the mitochondrial size. A correlation was found with smaller colonies exhibiting lower rates of oxygen consumption, and yeast labelled with fluorescent MitoTracker^TM consistently exhibited reduced mitochondrial activity. It appears that Casiopeína IIIEa gives rise to smaller yeast and petite-like colonies by reducing the mitochondrial respiratory activity without significantly affecting the mitochondrial structure.