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Endogenous ribonucleotides(RNs)and deoxyribonucleotides(dRNs)are important metabolites related to the pathogenesis of many diseases.In light of their physiological and pathological significances,a novel and sensitive pre-column derivatization method with N-(t-butyldimethylsilyl)-N-methyltri-fluoroacetamide(MTBSTFA)was developed to determine RNs and dRNs in human cells using high-performance liquid chromatography tandem mass spectrometry(HPLC-MS/MS).A one-step extraction of cells with 85％methanol followed by a simple derivatization reaction within 5 min at room temper-ature contributed to shortened analysis time.The derivatives of 22 nucleoside mono-,di-and tri-phosphates were retained on the typical Cig column and eluted by ammonium acetate and acetonitrile in 9 min.Under these optimal conditions,good linearity was achieved in the tested calibration ranges.The lower limit of quantitation(LLOQ)was determined to be 0.1-0.4 μM for the tested RNs and 0.001-0.1 μM for dRNs.In addition,the precision(CV)was＜15％and the RSD of stability was lower than 10.4％.Furthermore,this method was applied to quantify the endogenous nucleotides in human colorectal carcinoma cell lines HCT116 exposed to 10-hydroxycamptothecin.In conclusion,our method has proven to be simple,rapid,sensitive,and reliable.It may be used for specific expanded studies on intracellular pharmacology in vitro.
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In an effort to understand the molecular events contributing to the cytotoxicity activity of resveratrol (RSV), we investigated its effects on human lung adenocarcinoma epithelial cell line A549 at different concentrations. Cellular nucleoside metabolic profiling was determined by an established liquid chromatography-mass spectrometry method in A549 cells. RSV resulted in significant decreases and imbalances of deoxyribonucleoside triphosphates (dNTPs) pools suppressing subsequent DNA synthesis. Meanwhile, RSV at high concentration caused significant cell cycle arrest at S phase, in which cells required the highest dNTPs supply than other phases for DNA replication. The inhibition of DNA synthesis thus blocked subsequent progression through S phase in A549 cells, which may partly contribute to the cytotoxicity effect of RSV. However, hydroxyurea (HU), an inhibitor of RNR activity, caused similar dNTPs perturbation but no S phase arrest, finally no cytotoxicity effect. Therefore, we believed that the dual effect of high concentration RSV, including S phase arrest and DNA synthesis inhibition, was required for its cytotoxicity effect on A549 cells. In summary, our results provided important clues to the molecular basis for the anticancer effect of RSV on epithelial cells.
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Objective To evaluate thetherapeutic effect of sodium deoxyribonucleotide injection at Zusanli point for chemotherapy-induced leukopenia in patients with lung cancer.MethodsA total of 116 chemotherapy-induced leukopenia patients with lung cancer were randomly divided into a therapy group and a control group, 60 in the treatment group and 56 in the control group. The patients in the treatment group were treated with sodium deoxyribonucleotide injection atZusnlipoint, and those in the control group were treated with vein injection of sodium deoxyribonucleotide.Results The total effective rate in the treatment group was significant higher than that in the control group (91.7%vs.76.8%;χ2=4.890,P=0.032). The effective rates on days 3,5,7 and 10 in the treatment group were also significant higher than those in the control group (on day 3: 51.7%vs. 32.1%,χ2=4.530,P=0.036; on day 5: 76.7%vs. 53.6%,χ2=6.840,P=0.018; on day 7: 85.0%vs. 67.9%,χ2=4.770,P=0.026; on day 10: 78.3%vs. 53.6%,χ2=7.960,P=0.011).Conclusion Sodium deoxyribonucleotide injection atZusanlipoint has definite efficacy for chemotherapy-induced leukopenia in patients with lung cancer.
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AIM:To analyse sequences of p62 dok amino acid and cDNA and to investigate p62 dok tyrosine phosphorylation and its relation with p21 ras GAP. METHODS:The purified p62 dok was extracted from CHO/IR cells. The peptide sequence of p62 dok was carried out on a high performance analyzer. PCR was performed with the primers designed from the sequence of p62 dok amino acid. Western blot and immunoprecipitation were used to identify tyrosine phosphorylation of p62 dok and the binding of p62 dok with p21 ras GAP. RESULTS:The p62 dok cDNA is a 1863 bp sequence and code 481 amino acid with 15 tyrosine residues and a putative pleckstrin homology domain. The p62 dok protein is rich in PxxP motif. The tyrosine-phosphorylated p62 dok can bind p21 ras GAP. CONCLUSION:Perhaps p62 dok is a new signaling molecule and play an important role in insulin signaling networks through RAS/MAPK pathway.