The adduct formation between the thioguanine-polyamine ligands and DNA with the AP site under UVA irradiated and non-irradiated conditions

The AP sites are representative of DNA damage and known as an intermediate in the base excision repair (BER) pathway which is involved in the repair of damaged nucleobases by reactive oxygen species, UVA irradiation, and DNA alkylating agents. Therefore, it is expected that the inhibition or modulation of the AP site repair pathway may be a new type of anticancer drug. In this study, we investigated the effects of the thioguanine-polyamine ligands (^SG-ligands) on the affinity and the reactivity for the AP site under UVA irradiated and non-irradiated conditions. The ^SG-ligands have a photo-reactivity with the A-F-C sequence where F represents a tetrahydrofuran AP site analogue. Interestingly, the ^SG-ligands promoted the β-elimination of the AP site followed by the formation of a covalent bond with the β-eliminated fragment without UVA irradiation.     

Assessment of the chitosan-functionalized graphene oxide as a carrier for loading thioguanine,an antitumor drug and effect of urea on adsorption process: Combination of DFT computational and molecular dynamics simulation studies

In this study, the interaction thioguanine (TG) anticancer drug with the functionalized graphene oxide (GO) nanosheet surface is theoretically studied in both gas phase and separately in physiological media using the density functional theory (DFT) calculations. DFT calculations indicated the adsorption and solvation energies are negative for f-GONS/TG complexes which propose the adsorption process of TG molecule onto the f-GONS surface is possible from the energetic viewpoint. QTAIM calculations confirm the nature of partially covalent-partially electrostatic between drug and nanosheet. These results are sorely relevant that an approach for loading of TG molecule is the chemical modification of GO using covalent functionalization which can serve as a nanocarrier to load drug molecules. Moreover, to understand the effect of urea on the nature of the interaction between TG and f-GONS, molecular dynamics (MD) simulation was employed. The results indicated that in the presence of urea the adsorption process gets affected and leads to instability of system, while the affinity of the TG for adsorption onto GO surface is increased in pure water.

Communicated by Ramaswamy H. Sarma     

High-performance liquid chromatographic assay of metabolites of thioguanine and mercaptopurine in capillary blood

The main metabolites of the cytotoxic drugs thioguanine (6TG) and mercaptopurine (6MP) can be measured conveniently in red blood cells (RBC). Isolation of RBC, however, is laborious and requires some milliliters of blood. This HPLC assay allows measurements of thiopurine metabolites in very small blood samples obtained from the finger-tip. The metabolites, derivatives of 6TG and methylmercaptopurine (6MeMP), were extracted and hydrolized with perchloric acid to liberate the corresponding base. 6MeMP is completely transformed under these conditions to 4-amino-5-(methylthio)carbonyl imidazole. The chromatographic separation of 6TG and this imidazole was performed in a single run under isocratic conditions within 10 min using a 70 mm column. The quantification limit was 0.5 nmol/ml for 6TG and 3 nmol/ml blood for 6MeMP. The accuracy was 83% for 6TG (CV=3%) over the concentration range of 0.5-20 nmol/ml blood and 102% (CV=4%) for 6MeMP over the range of 3-150 nmol/ml blood. The intra-assay CV ranged from 5.4 to 7.4% for 6TG and from 6.2 to 10.6% for 6MeMP. The inter-assay CV was 7.5 and 9.5% in a pooled blood sample. The levels in RBC in whole blood were nearly coincident with those obtained in separated RBC, isolation of RBC therefore is not necessary for these measurements, if the drugs are given per os in the day before blood sampling. The concentration of 6MeMP nucleotides is more dependent on the given 6MP dose than the concentration of 6TG nucleotides. Intraindividual variations were small at unchanged drug doses, interindividual metabolite concentrations were highly variable.     

The regulatory effect of 6-TG on lncRNA–miRNA–mRNA ceRNA network in triple-negative breast cancer cell line

Breast cancer is one of the most prevalent and recurring cancer types that leads to deaths in women. Triple-negative breast cancer (TNBC) is difficult to treat due to the lack of therapeutic targets. Many studies have focused on identifying drugs for use as alternative treatments for breast cancer. Thioguanine (6-TG) exerts antitumor effects in cancer. Increasing evidence has demonstrated that competitive endogenous ribonucleic acids (ceRNAs) are involved in cancer processes. However, the mechanism by which 6-TG regulates lncRNA–miRNA–mRNAs has not been elucidated. We evaluated the antitumor effect of 6-TG in MDA-MB-231 cells and comprehensively analyzed the RNA-Seq data of MDA-MB-231 cells treated with 6-TG. Our results showed that most tumor pathways were blocked by 6-TG. The hub genes were FN1, FLNA, FLNB, VCL, GSN, MYH10, ACTN4, KDR and EREG, and they were all down-regulated after 6-TG treatment. The coexpression network consisted of 18 microRNAs (miRNAs), 9 long noncoding RNAs (lncRNAs) and 20 mRNAs. Hsa-mir-16-5p and Hsa-mir-335-5p targeted the greatest number of mRNAs in the network. These molecules could bind to PAX8-AS1 and eliminate the inhibition of target mRNA expression. We showed that PAX8-AS1 is the main lncRNA affected by 6-TG and that PAX8-AS1 regulates the hub genes in tumor pathways by competitively binding with miR-16-5p and miR-335-5p.     

Effect of O6-alkylguanine-DNA alkyltransferase on the frequency and spectrum of mutations induced by N-methyl-N''-nitro-N-nitrosoguanidine in the HPRT gene of diploid human fibroblasts

N-Methyl-N′-nitro-N-nitrosoguanidine (MNNG) reacts with 12 nucleophilic sites in DNA to induce a variety of lesions, but O⁶-methylguanine (O⁶-MeG) and O⁴-methylthymine are the most effective premutagenic lesions produced, mispairing with thymine and guanine, respectively. O⁶-MeG is repaired by O⁶-alkylguanine-DNA alkyltransferase (AGT), which removes the methyl group from the O⁶ position and transfers it to itself, rendering the transferase inactive. When diploid human fibroblasts were exposed to 25 μM, O⁶-benzylguanine (O⁶-BzG) in the medium for 3 h, their level of AGT activity was dramatically reduced, to a level of at most 1.6% of the control. Populations of cells pretreated with this level of O⁶-BzG for 2 h or not pretreated, were exposed to MNNG at a concentration of 2, 4 or 6 μM in the presence or absence of O⁶-BzG and assayed for survival of colony-forming ability and the frequency of 6-thioguanine-resistant cells (mutations induced in the HPRT gene). O⁶-BzG (25 μM) was also present in the appropriate half of the cells during the 24 h immediately follwing exposure to MNNG. This 27-h exposure to O⁶-BzG alone had no cytotoxic or mutagenic effect on the cells but significantly increased the cytotoxicity and mutagenecity of MNNG, increasing the mutant frequency to that found previously in human cells constitutively devoid of AGT activity. At doses of 2 μM and 4 μM MNNG, the mutant frequency observed with the AGT-depleted cells was 120 × 10⁻⁶ and 240 × 10⁻⁶, respectively; in the cells with abundant AGT activity, these values were 10 × 10⁻⁶ and 20 × 10⁻⁶, respectively. DNA-sequence analysis of the coding region of the HPRT gene in 36 independent mutants obtained from MNNG-treated AGT-depleted populations and 36 from the control populations showed that even though AGT repair lowered the frequency of mutants by more than 90%, it did not affect the kinds of mutations induced by MNNG nor the strand distribution of the premutagenic guanine lesions. In mutants from the AGT-depleted cells, there were 26 base substitutions and 13 putative splice site mutations; in the control, there were 25 base substitutions and 11 splice site mutations. All but two substitutions involved G · C with 92% being G · C → A · T. In both sets, of the premutagenic lesions were located in the nontranscribed strand. Many ‘hot spots’ were seen, and there was evidence that AGT repaired more lesions from the 5′ half of the gene than from the 3′ half.     

Purification and characterization of Escherichia coli xanthine-guanine phosphoribosyltransferase produced by plasmid pSV2gpt

The enzyme xanthine-guanine phosphoribosyltransferase from scherichia coli cells harboring the plasmid pSV2gpt has been purified 30-fold to near homogeneity by single-step GMP-agarose affinity chromatography. It has a K_m value of 2.5, 42 and 182 μM for the substrates guanine, xanthine and hypoxanthine, respectively, with guanine being the most preferred substrate. The enzyme exhibits a K_m value of 38.5 μM for PRib-PP with guanine as second substrate and of 100 μM when xanthine is used as the second substrate. It is markedly inhibited by 6-thioguanine, GMP and to a lesser extent by some other purine analogues. Thioguanine has been found to be the most potent inhibitor. The subunit molecular weight of xanthine-guanine phosphoribosyltransferase was determined to be 19 000. The in situ activity assay on a nondenaturing polyacrylamide gel electrophoresis gel has indicated that a second E. coli phosphoribosyltransferase preferentially uses hypoxanthine as opposed to guanine as a substrate, and it does not use xanthine.