guanosine-monophosphate and Breast-Neoplasms

The next-generation sequencing studies of breast cancer have reported that the tumour suppressor P53 (TP53) gene is mutated in more than 40% of the tumours. We studied the levels of oxidative lesions, including 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), along the coding strand of the exon 5 in breast cancer patients as well as in a reactive oxygen species (ROS)-attacked breast cancer cell line using the ligation-mediated polymerase chain reaction technique. We detected a significant 'in vitro' generation of 8-oxodG between the codons 163 and 175, corresponding to a TP53 region with high mutation prevalence, after treatment with xanthine plus xanthine oxidase, a ROS-generating system. Then, we evaluated the occurrence of oxidative lesions in the DNA-binding domain of the TP53 in the core needle biopsies of 113 of women undergoing breast investigation for diagnostic purpose. An increment of oxidative damage at the -G- residues into the codons 163 and 175 was found in the cancer cases as compared to the controls. We found significant associations with the pathological stage and the histological grade of tumours. As the major news of this study, this largest analysis of genomic footprinting of oxidative lesions at the TP53 sequence level to date provided a first roadmap describing the signatures of oxidative lesions in human breast cancer. Our results provide evidence that the generation of oxidative lesions at single nucleotide resolution is not an event highly stochastic, but causes a characteristic pattern of DNA lesions at the site of mutations in the TP53, suggesting causal relationship between oxidative DNA adducts and breast cancer.

Topics: Adult; Aged; Breast Neoplasms; Case-Control Studies; Cell Line, Tumor; Codon; DNA Adducts; DNA, Neoplasm; Exons; Female; Guanosine Monophosphate; Humans; Middle Aged; Oxidative Stress; Point Mutation; Tumor Suppressor Protein p53

The development of cancer and fibrotic diseases has been shown to be highly dependent on disregulation of cap-dependent translation. Binding protein eIF4E to N(7)-methylated guanosine capped mRNA has been found to be the rate-limiting step governing translation initiation, and therefore represents an attractive target for drug discovery. Our group has found that 7-benzyl guanosine monophosphate (7Bn-GMP) is a potent antagonist of eIF4E cap binding (K(d) = 0.8 μM). Recent X-ray crystallographic studies have revealed that the cap-dependent pocket undergoes a unique structural change in order to accommodate the benzyl group. Unfortunately, 7Bn-GMP is not cell permeable. Recently, we have prepared a tryptamine phosphoramidate prodrug of 7Bn-GMP, 4Ei-1, and shown that it is a substrate for human histidine triad nucleotide binding protein (hHINT1) and inhibits eIF4E initiated epithelial-mesenchymal transition (EMT) by Zebra fish embryo cells. To assess the intracellular uptake of 4Ei-1 and conversion to 7Bn-GMP by cancer cells, we developed a sensitive assay using LC-ESI-MS/MS for the intracellular quantitation of 4Ei-1 and 7Bn-GMP. When incubated with the breast cancer cell line MDA-231 or lung cancer cell lines H460, H383 and H2009, 4Ei-1 was found to be rapidly internalized and converted to 7Bn-GMP. Since oncogenic mRNAs are predicted to have the highest eIF4E requirement for translation, we carried out chemosensitization studies with 4Ei-1. The prodrug was found to chemosensitize both breast and lung cancer cells to nontoxic levels of gemcitabine. Further mechanistic studies revealed that the expressed levels of eIF4E were substantially reduced in cells treated with 4Ei-1 in a dose-dependent manner. The levels of eI4E could be restored by treatment with the proteasome inhibitor MG-132. Taken together, our results demonstrate that 4Ei-1 is likely to inhibit translation initiation by eIF4E cap binding by both antagonizing eIF4E cap binding and initiating eIF4E proteasomal degradation.

Topics: Breast Neoplasms; Cell Line, Tumor; Chromatography, High Pressure Liquid; Crystallography, X-Ray; Deoxycytidine; Eukaryotic Initiation Factor-4E; Gemcitabine; Guanosine Monophosphate; Humans; Lung Neoplasms; Models, Chemical; Proteasome Endopeptidase Complex; Spectrometry, Mass, Electrospray Ionization