Cai-Yun Wang

Comparison of two exploratory data analysis methods for classification of Phyllanthus chemical fingerprint: unsupervised vs. supervised pattern recognition technologies

In this study, unsupervised and supervised classification methods were compared for comprehensive analysis of the fingerprints of 26 Phyllanthus samples from different geographical regions and species. A total of 63 compounds were identified and tentatively assigned structures for the establishment of fingerprints using high-performance liquid chromatography time-of-flight mass spectrometry (HPLC/TOFMS). Unsupervised and supervised pattern recognition technologies including principal component analysis (PCA), nearest neighbors algorithm (NN), partial least squares discriminant analysis (PLS-DA), and artificial neural network (ANN) were employed. Results showed that Phyllanthus could be correctly classified according to their geographical locations and species through ANN and PLS-DA. Important variables for clusters discrimination were also identified by PCA. Although unsupervised and supervised pattern recognitions have their own disadvantage and application scope, they are effective and reliable for studying fingerprints of traditional Chinese medicines (TCM). These two technologies are complementary and can be superimposed. Our study is the first holistic comparison of supervised and unsupervised pattern recognition technologies in the TCM chemical fingerprinting. They showed advantages in sample classification and data mining, respectively.

Application of artificial neural network to investigate the effects of 5-fluorouracil on ribonucleotides and deoxyribonucleotides in HepG2 cells

Endogenous ribonucleotides and deoxyribonucleotides are essential metabolites that play important roles in a broad range of key cellular functions. Their intracellular levels could also reflect the action of nucleoside analogues. We investigated the effects of 5-fluorouracil (5-FU) on ribonucleotide and deoxyribonucleotide pool sizes in cells upon exposure to 5-FU for different durations. Unsupervised and supervised artificial neural networks were compared for comprehensive analysis of global responses to 5-FU. As expected, deoxyuridine monophosphate (dUMP) increased after 5-FU incubation due to the inhibition of thymine monophosphate (TMP) synthesis. Interestingly, the accumulation of dUMP could not lead to increased levels of deoxyuridine triphosphate (dUTP) and deoxyuridine diphosphate (dUDP). After the initial fall in intracellular deoxythymidine triphosphate (TTP) concentration, its level recovered and increased from 48 h exposure to 5-FU, although deoxythymidine diphosphate (TDP) and TMP continued to decrease compared with the control group. These findings suggest 5-FU treatment caused unexpected changes in intracellular purine polls, such as increases in deoxyadenosine triphosphate (dATP), adenosine-triphosphate (ATP), guanosine triphosphate (GTP) pools. Further elucidation of the mechanism of action of 5-FU in causing these changes should enhance development of strategies that will increase the anticancer activity of 5-FU while decreasing its resistance.

Profiling ribonucleotide and deoxyribonucleotide pools perturbed by gemcitabine in human non-small cell lung cancer cells

In this study, we investigated the dosage effect of gemcitabine, an inhibitor of ribonucleotide reductase (RR), on cellular levels of ribonucleotides and deoxyribonucleotides using high performance liquid chromatography-electrospray ionization tandem mass spectrometric method. As anticipated, after 4-h incubation of non-small cell lung cancer (A549) cells with gemcitabine at 0.5 and 2 μM, there were consistent reductions in levels of deoxyribonucleoside diphosphates (dNDP) and their corresponding deoxyribonucleoside triphosphates (dNTP). However, after 24-h exposure to 0.5 μM gemcitabine, the amounts of dNTP were increased by about 3 fold, whereas cells after 24-h 2 μM gemcitabine treatment exhibited deoxycytidine diphosphate (dCDP), deoxyadenosine diphosphate (dADP) and deoxyguanosine diphosphate (dGDP) levels less than 50% of control values, with deoxycytidine triphosphate (dCTP) and deoxyguanosine triphosphate (dGTP) returning to the control level. Using cell cycle analysis, we found that 24-h incubation at 0.5 μM gemcitabine resulted in a significant increase in S phase arrest, while 2 μM treatment increased G0/G1 population. Our data demonstrated the correlation between the level of RR and the increased levels of dNTPs in the group of 0.5 μM treatment for 24-h with a markedly reduced level of dFdCTP. Accordingly, we proposed that the dosage of dFdC could determine the arrested phase of cell cycle, in turn affecting the recovery of dNTPs pools.

Effect of Phyllanthus amarus Extract on 5-Fluorouracil-Induced Perturbations in Ribonucleotide and Deoxyribonucleotide Pools in HepG2 Cell Line.

The aim of this study was to investigate the antitumor activities of Phyllanthus amarus (PHA) and its potential of herb–drug interactions with 5-Fluorouracil (5-FU). Cell viability, ribonucleotides (RNs) and deoxyribonucleotides (dRNs) levels, cell cycle distribution, and expression of thymidylate synthase (TS) and ribonucleotide reductase (RR) proteins were measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, high performance liquid chromatography tandem mass spectrometry (HPLC/MS/MS) method, flow cytometry and Western blot analysis, respectively. Our standardized PHA extract showed toxicity to HepG2 cells at high concentrations after 72 h exposure and induced G2/M cell cycle arrest. Combined use of 5-FU with PHA resulted in significant decreases in ATP, CTP, GTP, UTP and dTTP levels, while AMP, CMP, GMP and dUMP levels increased significantly compared with use of 5-FU alone. Further, PHA could increase the role of cell cycle arrest at S phase induced by 5-FU. Although PHA alone had no direct impact on TS and RR, PHA could change the levels of RNs and dRNs when combined with 5-FU. This may be due to cell cycle arrest or regulation of key enzyme steps in intracellular RNs and dRNs metabolism.

Determination of corilagin in rat plasma using a liquid chromatography–electrospray ionization tandem mass spectrometric method

A sensitive and simple liquid chromatography-tandem mass spectrometric (HPLC-MS/MS) method for the determination of corilagin in rat plasma has been developed. Samples were prepared with protein precipitation method and analyzed with a triple quadrupole tandem mass spectrometer. We employed negative electrospray ionization as the ionization source and the analytes were detected in multiple reaction monitoring mode. Separation was achieved on a C8 column eluted with mobile phase consisting of methanol-0.1% formic acid in a gradient mode at the flow rate of 0.3 mL/min. The total run time was 7.0 min.This method was proved to have good linearity in the concentration range of 2.5-1000.0 ng/mL. The lower limit of quantification of corilagin was 2.5 ng/mL. The intra- and inter-day relative standard deviationa across three validation runs for four concentration levels were both <9.8%. The relative error was within ±6.0%. This assay offers advantages in terms of expediency and suitability for the analysis of corilagin in rat plasma. The practical utility of this new HPLC-MS/MS method was confirmed in pilot plasma concentration studies in rats following oral administration.

Quantitation of kudinoside A, kudinoside D and kudinoside F in human plasma using a high performance liquid chromatography–electrospray ionization tandem mass spectrometric method

A sensitive and selective high performance liquid chromatography-tandem mass spectrometric (HPLC-MS/MS) method for the simultaneous determination of kudinoside A, kudinoside D and kudinoside F in human plasma has been firstly developed. Samples were prepared after protein precipitation and analyzed on a C18 column interfaced with a triple quadrupole tandem mass spectrometer. Negative electrospray ionization was employed as the ionization source. The mobile phase consisted of acetonitrile-water (35:65) at the flow rate of 0.3mL/min. The analytes and internal standard Ginsenoside Rb1 were both detected by use of multiple reaction monitoring mode. The method was linear in the concentration range of 2.5-1000.0ng/mL. The lower limit of quantification (LLOQ) was 2.5ng/mL. The intra-and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 12.4%. The accuracy determined at three concentrations was within ±4.9% in terms of relative error. The total run time was 7.0min. This assay offers advantages in terms of expediency, and suitability for the analysis of kudinoside A, kudinoside D and kudinoside F in various biological fluids.

ZH-1 enhances the anticancer activity of gemcitabine via deoxyribonucleotide synthesis and apoptotic pathway against A549 cells

The purpose of this study was to investigate the inhibitory effect of ZH-1 ((6S,9aS,6aR,9bR)-6-(phenylcarbonyl)-6,6a,9a,9b-tetrahydro-8H-azolidino[3,4-a]b enzo [e]indolizine-7,9-dione) and its potential interaction with gemcitabine in A549 cells. MTT assay showed that the combined use of gemcitabine and ZH-1 presented a significant inhibition effect on A549 cell growth with the cell viability from 82.3 ± 5.6% to 51.0 ± 6.6%. The CI value was 0.60 suggesting a synergistic effect between these two drugs. HPLC-MS/MS data indicated that combined treatment with gemcitabine and ZH-1 induced a significant decrease in deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyguanosine triphosphate and deoxythymidine triphosphate levels compared with use of gemcitabine alone. Five RNs as well as seven dRNs were considered to be significantly contributive to the discrimination of samples. Furthermore, western blot analysis revealed that the combination treatment caused A549 cell apoptosis via the intrinsic pathway by up-regulating Bax/Bcl-2 ratio, activating caspase-9, caspase-3 and poly-ADP-ribose polymerase, and promoting caspase-7, caspase-9 and poly-ADP-ribose polymerase cleavage. Collectively, the combined treatment with gemcitabine and ZH-1 exerted a strong synergistic action on anticancer activity through growth inhibition, perturbations in ribonucleotides and deoxyribonucleotides and the activation of intrinsic apoptotic signaling pathway.

Profiling of ribonucleotides and deoxyribonucleotides pools in response to DNA damage and repair induced by methyl methanesulfonate in cancer and normal cells

The absolute and relative pool sizes of deoxyribonucleotides (dRNs) are essential in DNA replication fidelity, DNA damage and repair. We found in this study that although DNA damage induced by methyl methanesulfonate (MMS) seemed similar in cancer (HepG2) and normal (LO2) cells, more extensive alterations in ribonucleotides (RNs) and dRNs pools occurred in HepG2 cells indicating that HepG2 cells were more vigilant to DNA damage. After 10 h repair, RNs pools were still severely perturbed in LO2 cells. Compared to LO2 cells, deoxyribonucleotide triphosphates (dNTPs) pools in HepG2 cells elevated by more folds which could facilitate more efficient DNA repair and improve survival probability following DNA damage, although this should definitely lead to higher mutation rates. DNA repair was more efficient in HepG2 cells at S phase and it partly came to an end while DNA repair was still uncompleted in LO2 cells outside S phase. In conclusion, our results demonstrated that HepG2 and LO2 cells presented many differences in nucleotide metabolism, cell cycle checkpoints and DNA repair pathways in response to DNA damage, which could be potential targets for cancer treatment.

Exploring the Antitumor Mechanism of High-Dose Cytarabine through the Metabolic Perturbations of Ribonucleotide and Deoxyribonucleotide in Human Promyelocytic Leukemia HL-60 Cells

Despite the apparent clinical benefits of high-dose cytarabine (Ara-C) over lower dose Ara-C in acute myeloid leukemia (AML) therapy, the mechanism behind high-dose Ara-C therapy remains uncertain. In this study, a LC-MS-based method was carried out to investigate the metabolic alteration of ribonucleotide and deoxyribonucleotide in human promyelocytic leukemia cells (HL-60) after treatment with Ara-C to reveal its antitumor mechanism. The metabolic results revealed that four nucleotides (ATP, ADP, CDP, and dCTP) could be used as potential biomarkers indicating the benefit of high-dose Ara-C over lower dose Ara-C treatment. Combining metabolic perturbation and cell cycle analysis, we conjectured that, apart from the acknowledged mechanism of Ara-C on tumor inhibition, high-dose Ara-C could present a specific action pathway. It was suggested that the pronounced rise in AMP/ATP ratio induced by high-dose Ara-C can trigger AMP-activated protein kinase (AMPK) and subsequently Forkhead Box, class O (FoxO), to promote cell cycle arrest. Moreover, the significant decrease in CDP pool induced by high-dose Ara-C might further accelerate the reduction of dCTP, which then aggravates DNA synthesis disturbance. As a result, all of these alterations led to heightened tumor inhibition. This study provides new insight in the investigation of potential mechanisms in the clinical benefits of high-dose Ara-C in therapy for AML.

Quantitation of camellianin A in HepG2 cells using a high performance liquid chromatography-electrospray ionization tandem mass spectrometric method

The present study was designed to develop a sensitive and selective high performance liquid chromatography-tandem mass spectrometric method for the determination of Camellianin A in HepG2 cells. The extraction of Camellianin A was achieved using 15% trichloroacetic acid and then separated on a C18 column interfaced with a triple quadrupole tandem mass spectrometer in multiple reaction monitoring mode. The mobile phase was consisted of methanol-water (0.1% formic acid) (55 : 45, V/V). The total run time was 5.0 min. The method was linear in the concentration range of 0.25-250.0 ng·mL-1. The lower limit of quantification was 0.25 ng·mL-1. The intra- and inter-day relative standard deviations of entire concentration range were less than 9.3%. The proposed HPLC-MS/MS method was successfully applied to detect the intracellular concentration of Camellianin A in HepG2 cells.