Expression Profiling of Nucleotide Metabolism-Related Genes in Human Breast Cancer Cells After Treatment with 5-Fluorouracil

5-Fluorouracil (5-FU) is one of the most widely used drugs for treatment of cancers, including breast cancer that exhibits its anticancer activity by inhibiting DNA synthesis and also incorporated into DNA and RNA. The objective of this investigation was to find out the total nucleotide metabolism genes regulated by 5-FU in breast cancer cell line. The breast cancer cell line MCF-7 was treated with the drug 5-FU. To analyze the expression of genes, we have conducted the experiment using 1.7k and 19k human microarray slide and confirmed the expression of genes by semiquantitative reverse transcription-polymerase chain reaction. The expression of 44 genes involved in the nucleotide metabolism pathway was quantified. Of these 44 genes analyzed, transcription of 6 genes were upregulated and 9 genes were downregulated. Earlier studies revealed that the transcription of genes for key enzymes like thymidylate synthase, thymidinekinase, and dihydropyrimidine dehydrogenase are regulated by 5-FU. This study identified some novel genes like thioredoxin reductase, ectonucleotide triphosphate dephosphorylase, and CTP synthase are regulated by 5-FU. The data also reveal large-scale perturbation in transcription of genes not involved directly in the known mechanism of action of 5-FU.

Inhibition of replication of rinderpest virus by 5-fluorouracil

5-Fluorouracil (5FU), an analogue of uracil, was found to inhibit the production of infectious particles of rinderpest virus (RPV) in Vero cells (African green monkey kidney cells) by 99%, at a concentration of 1 μg/ml. The levels of individual mRNA specific for five of the virus genes were also reduced drastically, while the level of mRNA for a cellular housekeeping gene—glyceraldehyde-3-phosphate dehydrogenase (GAPDH)—was unaltered by fluorouracil treatment of infected cells. Both virus RNA and protein synthesis showed inhibition in a dose-dependent manner. The virions which budded out of 5-fluorouracil-treated cells also contained reduced amounts of virus proteins compared with virus particles from untreated cells.

5-Fluorouracil induces structural changes in rat liver tRNA

5-fluorouracil (FUra) has been shown to modulate the aminoacylation function of rat liver tRNA. The present study was aimed at studying the structure-function relationship of FUra-substituted tRNA. Male Wistar rats (2-3 month old) were given a single i.p. injection of FUra at 50, 250, or 500 mg/kg body wt. and FUra-substituted total liver tRNA, i.e. tRNA(FUra50, 250, and 500, respectively, were isolated 3 h later. Normal tRNA (tRNA(N)) was isolated from saline-treated control rats. Thermal denaturation studies showed higher melting temperatures for tRNA(FUra) compared to tRNA(N). Heat denaturation followed by renaturation of total tRNA did not affect the activity of tRNA(N) and tRNA(FUra50), where as tRNA(FUra250 and 500) lost 35% and 72% of activity, respectively, compared to the corresponding group of non-denatured tRNA. Antibodies specific to rat liver tRNA recognized normal and FUra-substituted tRNA in the order of tRNA(N) > tRNA(FUra50) > or = tRNA(FUra250) > tRNA(FUra500) in an avidin-biotin micro-enzyme linked immunosorbant assay. tRNA(N) or tRNA(FUra50) preincubated with tRNA antiserum showed 74% and 59% of aminoacylation activity, respectively, compared to that of corresponding tRNA preincubated with normal rabbit IgG. However, activities of similarly treated tRNA(FUra250 and 500) were not affected. The observations of possible changes in the secondary structure of rat liver tRNA upon incorporation of FUra are discussed.

Altered ultrastructure of rinderpest virus and its nucleocapsids induced by 5-fluorouracil

he ultrastructure of purified rinderpest virus and intracellular viral nucleocapsids from infected vero cells treated with a subtoxic dose of 5-fluorouracil (5-Fu) (1 mug/ml), has been analysed by transmission electron microscopy, and compared with that of normal virus particle and nucleocapsids. The results reveal dramatic alterations in the structure of both virions and nucleocapsids. The surface glycoprotein projection of virions was not seen or present at a much reduced level. The intracellular nucleocapsids showed pronounced structural changes,with respect to size, shape and fine structure. The length of treated nucleocapsids is much smaller as compared to the control. The central hollow core is missing in case of drug-treated nucleocapsid and the herring bone structure is replaced by a 'beads on string' structure. The presence of N protein, which is a major structural component of nucleocapsids was seen in 5-Fu-treated cells, but it was associated with a predominantly diffused form of nucleocapsids as seen by immunoelectron microscopy. We report here the first definitive and visual evidence of altered structure of virions and their nucleocapsids after 5-Fu treatment

Thymidine inhibits the incorporation of 5-fluoro-2'-deoxyuridine to DNA of mouse mammary tumour

5-Fluoro-2'-deoxyuricine is incorporated into DNA of mouse breast tumour Image . The incorporation is inhibited by thymidine. Part of the fluorodeoxyuridine is cleaved to fluorouracil and is incorporated into RNA. This incorporation is enhanced by thymidine. The result suggests that the major mechanism of action of the fluorouracil is due to its incorporation into RNA. FUra, 5-Fluorouracil; FdUR, 5-Fluoro-2'-deoxyuridine; FdUMP, 5-Fluoro-2'-deoxyuridine-5'-monophosphate.

Investigation of biodegradable and biocompatible castor oil poly(mannitol-citric-sebacate) polyester as a drug carrier

Castor oil-based poly(mannitol-citric sebacate) was synthesized by simple, catalyst-free melt condensation process using monomers having potential to be metabolized in vivo. The polymer was characterized using various techniques and the tensile and hydration properties of the polymers were also determined. The biocompatibility of the polymer was tested using human foreskin fibroblasts cells. The in vitro degradation studies show that the time for complete degradation of the polymer was more than 21 days. The usage of castor oil polyester as a drug carrier was analysed by doping the polymer with 5-fluorouracil model drug and the release rate was studied by varying the percentage loading of drugs and the pH of the PBS solution medium. The cumulative drug-release profiles exhibited a biphasic release with an initial burst release and cumulative 100% release within 42 h. To understand the role of the polymer as a drug carrier in the release behaviour, drug-release studies were conducted with another drug, isoniazid. The release behaviour of isoniazid drug from the same polymer matrix followed an nth order kinetic model and 100% cumulative release was achieved after 12 days. The variation in the release behaviour for two model drugs from the same polymer matrix suggests a strong interaction between the polymer and the drug molecule.

Dual Drug Conjugate Loaded Nanoparticles for the Treatment of Cancer

Two antineoplastic agents, Imatinib (IM) and 5-Fluorouracil (FU) were conjugated by hydrolysable linkers through an amide bond and entrapped in polymeric Human Serum Albumin (HSA) nanoparticles. The presence of dual drugs in a common carrier has the advantage of reaching the site of action simultaneously and acting at different phases of the cell cycle to arrest the growth of cancer cells before they develop chemoresistance. The study has demonstrated an enhanced anticancer activity of the conjugate, and conjugate loaded stealth HSA nanoparticles (NPs) in comparison to the free drug in A-549 human lung carcinoma cell line and Zebra fish embryos (Danio rerio). Hydrolysability of the conjugate has also been demonstrated with complete hydrolysis being observed after 12 h. In vivo pharmacodynamics study in terms of tumor volume and pharmacokinetics in mice for conjugate (IM-SC-FU) and conjugate loaded nanoparticles showed significant anti-cancer activity. The other parameters evaluated were particle size (86nm), Poly Dispersive Index (PDI) (0.209), zeta potential (-49mV), drug entrapment efficiency (96.73%) and drug loading efficiency (89%). Being in stealth mode gives the potential for the NPs to evade Reticulo-Endothelial system (RES), achieve passive targeting by Enhanced Permeation Retention (EPR) effect with controlled release of the therapeutic agent. As the conjugate cleaves into individual drugs in the tumor environment, this promises better suppression of cancer chemoresistance by delivering dual drugs with different modes of action at the same site, thereby synergistically inhibiting the growth of cancerous tissue.