Effects of flavonoids derived from Taxus yunnanensis on p-glycoprotein and cytochrome P450 3A4

AbstractThe intestinal uptake of paclitaxel is hampered by trans-membrane efflux transporters such as P-glycoprotein (P-gp), and paclitaxel is mainly metabolized by cytochrome P450 3A4 (CYP3A4) presented in the liver. Our previous results demonstrated that flavonoids extracted from Taxus yunnanensis could improve the oral absorption of paclitaxel. The current study was purposed to investigate the effects of the flavonoid extracts on P-gp and CYP3A4 in vitro. The expression and activity of P-gp were detected by western blotting and intracellular rhodamine 123 accumulation assay in Caco-2 cells treated with the flavonoids extract. The expression of CYP3A4 was investigated by western blotting in mouse primary hepatocytes and the activity of CYP3A4 was detected by LC-MS/MS method using rat liver microsomes. Our results showed that the flavonoid extracts from T. yunnanensis could inhibit P-gp activity and concurrently decrease the expression and activity of CYP3A4. In conclusion, activity of P-gp and CYP3A4 could be inhibited by flavonoids extracted from T. yunnanensis which might be potential candidates for development of oral formulation of paclitaxel

Green Tea Polyphenols Protect against Acetaminophen-Induced Liver Injury by Regulating the Drug Metabolizing Enzymes and Transporters

Green tea polyphenols (GTPs) have been shown to exhibit diverse beneficial effects against a variety of diseases. Acetaminophen (APAP) overdose is one of the most frequent causes of drug-induced liver injury. In the current study, we aimed to investigate the protective effect of GTP on APAP-induced liver injury in mice and the underlying mechanisms involved. Male C57BL/6J mice were treated orally with different doses of GTP (37.5, 75, or 150 mg/kg) 4 h after APAP overdose (400 mg/kg) and continuously given every 8 h until sacrificed at 4, 12, 20, and 48 h after the first treatment of GTP. Survival rate and histological and biochemical assessments were performed to evaluate the APAP-induced liver injury. Protein expression of multiple drug metabolizing enzymes and transporters was measured to demonstrate the possible mechanisms involved. Our results revealed that administration of different doses of GTP significantly alleviated APAP-induced liver injury by improving the survival rate, hepatocellular necrosis, and ALT/AST/GSH levels after APAP overdose (400 mg/kg). The protein expression of APAP-induced drug transporters and metabolizing enzymes was mostly induced by GTP treatment, which was followed by reduction in drug transporters at the later time points. The current study collectively demonstrated that GTP protects against APAP-induced liver injury, possibly through regulating drug metabolizing enzymes and transporters after APAP overdose

Use of recombinant microRNAs as antimetabolites to inhibit human non-small cell lung cancer.

During the development of therapeutic microRNAs (miRNAs or miRs), it is essential to define their pharmacological actions. Rather, miRNA research and therapy mainly use miRNA mimics synthesized in vitro. After experimental screening of unique recombinant miRNAs produced in vivo, three lead antiproliferative miRNAs against human NSCLC cells, miR-22-3p, miR-9-5p, and miR-218-5p, were revealed to target folate metabolism by bioinformatic analyses. Recombinant miR-22-3p, miR-9-5p, and miR-218-5p were shown to regulate key folate metabolic enzymes to inhibit folate metabolism and subsequently alter amino acid metabolome in NSCLC A549 and H1975 cells. Isotope tracing studies further confirmed the disruption of one-carbon transfer from serine to folate metabolites by all three miRNAs, inhibition of glucose uptake by miR-22-3p, and reduction of serine biosynthesis from glucose by miR-9-5p and -218-5p in NSCLC cells. With greater activities to interrupt NSCLC cell respiration, glycolysis, and colony formation than miR-9-5p and -218-5p, recombinant miR-22-3p was effective to reduce tumor growth in two NSCLC patient-derived xenograft mouse models without causing any toxicity. These results establish a common antifolate mechanism and differential actions on glucose uptake and metabolism for three lead anticancer miRNAs as well as antitumor efficacy for miR-22-3p nanomedicine, which shall provide insight into developing antimetabolite RNA therapies

Endometrium metabolomic profiling reveals potential biomarkers for diagnosis of endometriosis at minimal-mild stages

Abstract Background The sensitivity and specificity of non-invasive diagnostic methods for endometriosis, especially at early stages, are not optimal. The clinical diagnostic indicator cancer antigen 125 (CA125) performs poorly in the diagnosis of minimal endometriosis, with a sensitivity of 24%. Therefore, it is urgent to explore novel diagnostic biomarkers. We evaluated the metabolomic profile variation of the eutopic endometrium between minimal-mild endometriosis patients and healthy women by ultra-high-performance liquid chromatography coupled with electrospray ionization high-resolution mass spectrometry (UHPLC-ESI-HRMS). Methods Our study comprised 29 patients with laparoscopically confirmed endometriosis at stages I-II and 37 infertile women who underwent diagnostic laparoscopy combined with hysteroscopy from January 2014 to January 2015. Eutopic endometrium samples were collected by pipelle endometrial biopsy. The metabolites were quantified by UHPLC-ESI-HRMS. The best combination of biomarkers was then selected by performing step-wise logistic regression analysis with backward elimination. Results Twelve metabolites were identified as endometriosis-associated biomarkers. The eutopic endometrium metabolomic profile of the endometriosis patients was characterized by a significant increase in the concentration of hypoxanthine, L-arginine, L-tyrosine, leucine, lysine, inosine, omega-3 arachidonic acid, guanosine, xanthosine, lysophosphatidylethanolamine and asparagine. In contrast, the concentration of uric acid was decreased. Metabolites were filtered by step-wise logistic regression with backward elimination, and a model containing uric acid, hypoxanthine, and lysophosphatidylethanolamine was constructed. Receiver-operating characteristic (ROC) analysis confirmed the prognostic value of these parameters for the diagnosis of minimal/mild endometriosis with a sensitivity of 66.7% and a specificity of 90.0%. Conclusions Metabolomics analysis of the eutopic endometrium in endometriosis was effectively characterized by UHPLC-ESI-HRMS-based metabolomics. Our study supports the importance of purine and amino acid metabolites in the pathophysiology of endometriosis and provides potential biomarkers for semi-invasive diagnosis of early-stage endometriosis

Schisandra sphenanthera Extract Facilitates Liver Regeneration after Partial Hepatectomy in Mice

ABSTRACT Liver regeneration after surgical liver resection is crucial for the restoration of liver mass and the recovery of liver function. Schisandra sphenanthera extract (Wuzhi tablet, WZ) is a preparation of an extract from the dried ripe fruit of Schisandra sphenanthera Rehd. et Wils, a traditional hepatoprotective herb. Previously, we found that WZ could induce liver regeneration-related genes against acetaminopheninduced liver injury. However, whether WZ can directly facilitate liver regeneration after liver resection remains unknown. We investigated whether WZ has potential in promoting liver regeneration after a partial hepatectomy (PHX) in mice. Remnant livers were collected 1, 1.5, 2, 3, 5, 7, and 10 days after PHX. Hepatocyte proliferation was assessed using the Ki-67 labeling index. Western blot analysis was performed on proteins known to be involved in liver regeneration. The results demonstrated that WZ significantly increased the liver-to-body weight ratio of mice after PHX but had no effect on that of mice after a sham operation. Additionally, the peak hepatocyte proliferation was observed at 1.5 days in PHX/WZ-treated mice but at 2 days in PHX/saline-treated mice, as evidenced by the Ki-67 positive ratio. Furthermore, WZ significantly increased the protein expression of ligand-induced phosphorylation of epidermal growth factor receptor and up-regulated cyclin D1, cyclin D-dependent kinase 4, phosphorylated retinoblastoma, and proliferating cell nuclear antigen protein expression and down-regulated the expression of cell cycle inhibitors p21 and p27 in the regenerative process after PHX. These results demonstrate that WZ significantly facilitates hepatocyte proliferation and liver regeneration after PHX

Determination of CH330331, a novel 4-anilinoquinazoline inhibitor of epidermal growth factor receptor tyrosine kinase, in human Caco-2 monolayers by high performance liquid chromatography with ultraviolet detection : application to a trans-epithelial transport study

4-Anilinoquinazolines (e.g. Iressa and Glivec) are a class of epidermal growth factor receptor tyrosine kinase (EGFR-TK) inhibitors widely used to treat non-small cell lung cancer and other tumors. However, low clinical response rate, resistance, and host toxicity of currently available EGFR-TK inhibitors prompt the development of second generation of TK inhibitors with improved efficacy, selectivity, and less resistance. CH330331 is a recently synthesized novel 4-anilinoquinazoline analog with confirmed anticancer activity in vitro and in vivo. To predict its oral pharmacokinetic behavior and transport nature in the intestine before entering clinical trials, we have developed and validated a high performance liquid chromatographic (HPLC) method for the determination of CH330331 in Caco-2 (a human colon cancer cell line) monolayers. The developed HPLC method was sensitive and reliable, with acceptable accuracy (90-110% of nominal values) and precision (intra- and inter-assay R.S.D. < 10%). The total running time was within 10 min, with acceptable separation of the target analytes. The lower limit of quantitation (LLOQ) value for CH330331 was 200 ng/ml when an aliquot of 100 μl sample was injected onto the HPLC. The validated HPLC method was applied to characterize the epithelial transport of CH330331 in Caco-2 monolayers. The transport of CH330331 across the Caco-2 monolayers from the apical to basolateral side was 8- to 10-fold higher than that from the basolateral to apical side. Co-incubation of sodium azide or MK-571, but not verapamil, significantly inhibited the apical to basolateral transport of CH330331. These findings provide initial evidence that the intestinal absorption of CH330331 is mediated by an active mechanism. Further studies are required to explore the interaction of CH330331 with ATP-binding cassette transporters and the possible influence on its pharmacokinetics and pharmacodynamic