RNA sequencing revealed the multi-stage transcriptome transformations during the development of gallbladder cancer associated with chronic inflammation.

Gallbladder cancer (GBC) is a highly malignant tumor with extremely poor prognosis. Previous studies have suggested that the carcinogenesis and progression of GBC is a multi-stage and multi-step process, but most of them focused on the genome changes. And a few studies just compared the transcriptome differences between tumor tissues and adjacent noncancerous tissues. The transcriptome changes, relating to every stage of GBC evolution, have rarely been studied. We selected three cases of normal gallbladder, four cases of gallbladder with chronic inflammation induced by gallstones, five cases of early GBC, and five cases of advanced GBC, using next-generation RNA sequencing to reveal the changes in mRNAs and lncRNAs expression during the evolution of GBC. In-depth analysis of the sequencing data indicated that transcriptome changes from normal gallbladder to gallbladder with chronic inflammation were distinctly related to inflammation, lipid metabolism, and sex hormone metabolism; transcriptome changes from gallbladder with chronic inflammation to early GBC were distinctly related to immune activities and connection between cells; and the transcriptome changes from early GBC to advanced GBC were distinctly related to transmembrane transport of substances and migration of cells. Expression profiles of mRNAs and lncRNAs change significantly during the evolution of GBC, in which lipid-based metabolic abnormalities play an important promotive role, inflammation and immune activities play a key role, and membrane proteins are very highlighted molecular changes.

Long-term exposure to genistein inhibits the proliferation of gallbladder cancer by downregulating the MCM complex.

Soy isoflavones are natural tyrosine kinase inhibitors closely associated with decreased morbidity and mortality of various tumors. The activation of tyrosine kinases such as ERBB2 is the mechanism by which cholecystitis transforms into gallbladder cancer (GBC), therefore, it is important to investigate the relationship between long-term exposure to soy isoflavones and the occurrence and progression of GBC. This case-control study (n = 85 pairs) found that the high level of plasma soy isoflavone-genistein (GEN) was associated with a lower risk of gallbladder cancer (≥326.00 ng/mL compared to ≤19.30 ng/mL, crude odds ratio 0.15, 95% CI 0.04-0.59; P for trend = 0.016), and that the level of GEN exposure negatively correlated with Ki67 expression in GBC tissue (n = 85). Consistent with these results, the proliferation of GBC cells was inhibited in the long-term exposure models of GEN in vitro and in vivo. The long-term exposure to GEN reduced the tyrosine kinase activity of ERBB2 and impaired the function of the PTK6-AKT-GSK3ß axis, leading to downregulation of the MCM complex in GBC cells. In summary, long-term exposure to GEN associated with soy products intake might play a certain role in preventing GBC and even inhibiting the proliferation of GBC cells.

The expression of growth differentiation factor 15 in gallbladder carcinoma.

PURPOSE: This study initially explored the expression of GDF-15 in gallbladder carcinoma and its clinical significance, and analyzed the correlation between the expression of GDF-15 and the clinicopathological features as well as the prognosis of patients with gallbladder carcinoma. METHODS: Enzyme-linked immunosorbent assay (ELISA) was used to determine the expression of GDF-15 in the serum of 42 patients with gallbladder cancer. The control group included 24 patients with cholecystitis and 20 healthy volunteers. The immunohistochemical method (IHC) was used to detect the expression of GDF-15 in 42 cases of gallbladder tumor tissue and 35 cases of adjacent non-tumor gallbladder tissue specimens. RESULTS: The results of ELISA showed that the concentration of GDF-15 in serum was considerably higher in gallbladder cancer patients than that in gallbladder benign lesions and healthy volunteers (p=0.006, p<0.001). In the group of patients with gallbladder cancer, the consistence of GDF-15 in patients with lymph node metastasis was significantly higher than that of patients without lymph node metastasis (p<0.001). Immunohistochemical staining showed that the expression of GDF-15 in gallbladder carcinoma was markedly higher than that in non-tumor gallbladder tissues (p=0.003), and the high expression of GDF-15 was significantly correlated with the differentiation grade of gallbladder carcinoma and tumor TNM stage (p=0.005, p=0.002). CONCLUSION: GDF-15 is related to the occurrence and development of gallbladder cancer. GDF-15 in serum can be used as a potential marker for the diagnosis of gallbladder cancer and can be used to predict the lymph node metastasis of gallbladder cancer.

Pharmacokinetics, bioavailability and metabolism of cligosiban, an antagonist of oxytocin receptor, in rat by liquid chromatography hyphenated with electrospray ionization tandem mass spectrometry.

Cligosiban is a highly-affinity nonpeptide oxytocin receptor antagonist. In this study, a simple an sensitive LC-MS/MS method was developed and validated for the determination of cligosiban in rat plasma. The plasma samples were pretreated with acetonitrile as precipitant and then separated on an ACQUITY BEH C18 column (2.1 × 50 mm, 1.7 µm) with 0.1% formic acid in water and acetonitrile as mobile phase. The analytes were monitored using selected reaction monitoring (SRM) mode with transitions at m/z 420.1→248.1 for cligosiban and m/z 304.1→161.1 for IS. The developed method showed good linearity over the concentration range of 1-1000 ng/mL with coefficient of correlation > 0.996. The lower limit of quantification (LLOQ) is 1 ng/mL. The method was validated for selectivity, precision, accuracy, recovery, and stability in accordance with FDA's guidance. The validated assay has been successfully applied to the pharmacokinetic study of cligosiban in rat plasma after intravenous and oral administration. According to the current results, the oral bioavailability of cligosiban was 63.82%. Furthermore, the metabolites present in rat liver microsomes (RLM), human liver microsomes (HLM) and rat plasma were analyzed by UHPLC-LTQ-Orbitrap-MS method, and four metabolites structurally identified based on their accurate masses, and fragment ions. The proposed metabolic pathways of cligosiban were demethylation and glucuronidation. This study is the first report on the pharmacokinetic and metabolic information of cligosiban, which would provide insights into the effectiveness and toxicity of cligosiban.