Comprehensive characterization of the <i>in vitro</i> and <i>in vivo</i> metabolites of geniposide in rats using ultra-high-performance liquid chromatography coupled with linear ion trap–Orbitrap mass spectrometer

<p>1. Geniposide (genipin 1-<i>O-</i>glucose), one of the major bioactive constituents isolated from Fructus Gardeniae, possesses many biological activities. In this study, an efficient strategy was developed using ultra-high-performance liquid chromatography coupled with linear ion trap–Orbitrap mass spectrometer (UPLC–LTQ–Orbitrap) to profile the <i>in vitro</i> and <i>in vivo</i> metabolic patterns of geniposide in rat liver microsomes (RLMs), plasma, urine, and various tissues. And post-acquisition data-mining methods including extracted ion chromatogram (EIC), multiple mass defect filters (MMDF), fragment ion searching (FISh), and isotope pattern filtering (IPF) were adopted to characterize the known and unknown metabolites.</p> <p>2. A total of 33 metabolites were detected and interpreted according to accurate mass measurement, diagnostic fragment ions, relevant drug biotransformation knowledge, and bibliography data. Among them, 17 metabolites were detected in the plasma, 31 metabolites were identified in the urine, six metabolites could be found in rat heart, 12 in liver, three in spleen, six in lung, 12 in kidney, six in brain, and four in RLMs.</p> <p>3. A series of corresponding reactions such as hydrolysis, hydroxylation, taurine conjugation, hydrogenation, decarboxylation, demethylation, sulfate conjugation, cysteine S-conjugation, glucosylation, and their composite reactions were all discovered.</p> <p>4. The results could provide comprehensive insights and guidance for elucidation of side effect mechanism and safety monitoring as well as for rational formulation design in drug delivery system. The newly discovered geniposide metabolites could be targets for future metabolism studies on the important chemical constituents from herbal medicines.</p

Association of Promoter Methylation of <i>RUNX3</i> Gene with the Development of Esophageal Cancer: A Meta Analysis

<div><p>Background</p><p>Runt-related transcription factor 3 (<i>RUNX3</i>) is a member of the runt-domain family of transcription factors. Emerging evidence indicates that RUNX3 is a tumor suppressor gene in several types of human cancers including esophageal cancer. However, the association between <i>RUNX3</i> promoter methylation and esophageal cancer remains unclear. Here we conducted a systematic review and meta-analysis to quantitatively evaluate the effects of <i>RUNX3</i> promoter methylation on the incidence of esophageal cancer.</p><p>Methods</p><p>A detailed literature search was made on Medline, Pubmed and Web of Science for related research publications written in English and/or Chinese. Methodological quality of the studies was also evaluated. The data were extracted and assessed by two reviewers independently. Analysis of pooled data were performed, the odds ratios (OR) were calculated and summarized respectively.</p><p>Results</p><p>Final analysis of 558 patients from 9 eligible studies was performed. The result showed that <i>RUNX3</i> methylation was significantly higher in esophageal cancer than in normal squamous mucosa from the proximal resection margin or esophageal benign lesions (OR = 2.85, CI = 2.01–4.05, P<0.00001). The prevalence of lymph node involvement, tumor size (T1–T2 vs T3–T4) and histological grade was significantly greater in <i>RUNX3</i>-negative cases (<i>RUNX3</i> unmethylated groups) than in <i>RUNX3</i>-positive cases (OR = 0.25, CI = 0.14–0.43, P<0.00001). <i>RUNX3</i> methylation was significantly higher in esophageal adenocarcinoma (EAC) than Barrett’s esophagus (OR = 0.35, CI = 0.20–0.59, P<0.0001). In addition, the pooled HR for overall survival (OS) showed that decreased <i>RUNX3</i> expression was associated with worse survival in esophageal cancer (HR = 4.31, 95% CI = 2.57–7.37, P<0.00001).</p><p>Conclusions</p><p>The results of this meta-analysis suggest that <i>RUNX3</i> methylation is associated with an increased risk, progression as well as worse survival in esophageal cancer. <i>RUNX3</i> methylation, which induces the inactivation of <i>RUNX3</i> gene, plays an important role in esophageal carcinogenesis.</p></div

The funnel plots were largely symmetric suggesting there were no publication biases in the meta-analysis of <i>RUNX3</i> methylation/expression and clinicopathological features as well as overall survival respectively.

<p>The funnel plot from 6 studies comparing esophageal cancers and normal squamous mucosa (A). The funnel plot from 4 studies in determining <i>RUNX3</i> hypermethylation in advanced stage (T3–T4) and early stage (T1–T2) (B). The funnel plot from 2 studies in determining <i>RUNX3</i> hypermethylation in Barrett’s esophagus (BE) and esophageal adenocarcinoma (EAC) (C). The funnel plot from 4 studies in determining the relationship between <i>RUNX3</i> hypermethylation and overall survival (OS) in esophageal cancer (D).</p

Flow chart of study selection.

<p>Flow chart of study selection.</p

All four included studies estimated the relationship between OS and <i>RUNX3</i> methylation/expression.

<p>The pooled HR for OS showed that decreased <i>RUNX3</i> expression was associated with worse survival in esophageal cancer, HR = 4.31, 95% CI = 2.57–7.37, P<0.00001.</p

<i>RUNX3</i> methylation significantly higher in esophageal adenocarcinoma (EAC) than Barrett’s esophagus (BE), OR = 0.35, CI = 0.20–0.59, P<0.0001.

<p><i>RUNX3</i> methylation significantly higher in esophageal adenocarcinoma (EAC) than Barrett’s esophagus (BE), OR = 0.35, CI = 0.20–0.59, P<0.0001.</p

Forest plot of odds ratio (OR) in 263 patients pooled in 4 studies in serum/cancer tissues DNA from advanced stage, including tumor size (T1–T2 vs T3–T4), lymph node involvement, lymph and blood vessels metastasis, and recurrence in esophageal carcinomas.

<p>The prevalence of lymph node involvement, tumor size (T1–T2 vs T3–T4) and histological grade was significantly greater in <i>RUNX3</i>-negative cases (<i>RUNX3</i> unmethylated group) than in <i>RUNX3</i>-positive cases, OR = 0.25, CI = 0.14–0.43, P<0.00001.</p

The pooled OR from 6 studies including 347 esophageal cancers and 246 normal squamous mucosa OR = 2.85, CI = 2.01–4.05, P<0.00001.

<p>The pooled OR from 6 studies including 347 esophageal cancers and 246 normal squamous mucosa OR = 2.85, CI = 2.01–4.05, P<0.00001.</p