Novel role of PAF1 in attenuating radiosensitivity in cervical cancer by inhibiting IER5 transcription.

BACKGROUND: Radiosensitivity is limited in cervical cancer (CC) patients due to acquired radiation resistance. In our previous studies, we found that immediate-early response 5 (IER5) is upregulated in CC cells upon radiation exposure and decreases cell survival by promoting apoptosis. The details on the transcriptional regulation of radiation-induced IER5 expression are unknown. Studies in recent years have suggested that Pol II-associated factor 1 (PAF1) is a pivotal transcription factor for certain genes "induced" during tumor progression. In this study, we investigated the role of PAF1 in regulating IER5 expression during CC radiotherapy. METHODS: PAF1 expression in CC cells was measured by western blotting, immunohistochemistry, and qRT-PCR, and the localization of PAF1 and IER5 was determined by immunofluorescence. The effect of PAF1 and IER5 knockdown by siRNA in Siha and Hela cells was studied by western blotting, qRT-PCR, CCK-8 assay, and flow cytometry. The physical interaction of PAF1 with the IER5 promoter and enhancers was confirmed using chromatin immunoprecipitation and qPCR with or without enhancers knockout by CRISPR/Cas9. RESULTS: We confirmed that PAF1 was highly expressed in CC cells and that relatively low expression of IER5 was observed in cells with highly expressed PAF1 in the nucleus. PAF1 knockdown in Siha and Hela cells was associated with increased expression of IER5, reduced cell viability and higher apoptosis rate in response to radiation exposure, while simultaneous PAF1 and IER5 knockdown had little effect on the proportion of apoptotic cells. We also found that PAF1 hindered the transcription of IER5 by promoting Pol II pausing at the promoter-proximal region, which was primarily due to the binding of PAF1 at the enhancers. CONCLUSIONS: PAF1 reduces CC radiosensitivity by inhibiting IER5 transcription, at least in part by regulating its enhancers. PAF1 might be a potential therapeutic target for overcoming radiation resistance in CC patients.

The Tissue Distribution of Four Major Coumarins after Oral Administration of Angelicae Pubescentis Radix Extract to Rats Using Ultra-High-Performance Liquid Chromatography.

Angelicae pubescentis radix (APR) is widely applied in treating rheumatoid arthritis in China. Coumarins are the major active compounds of APR extract including columbianetin, columbianetin acetate, osthole, and columbianadin. The in vivo behavior of the four major coumarins of APR has not been systematically reported. A feasible and reliable ultra-performance liquid chromatography (UPLC) method was established and validated for the quantification of the above four coumarins in rat various tissues (including heart, liver, spleen, lung, kidney, uterus, ovary, and muscle) after oral administration of APR extract. The separation was implemented on a Waters ACQUITY BEH C18 column (4.6 mm × 100 mm, 1.7 µm) with gradient mobile phase comprising acetonitrile-water (with 1mM formic acid) at a flow rate of 0.3 mL/min. The tissue homogenate samples were prepared by liquid-liquid extraction with ethyl acetate. The calibration curves were linear in the range of 1.6-20000 ng/mL for four coumarins with the lower limit of quantitation of 1.6 ng/mL in rat tissues. The intraday and interday precisions and recoveries were all within 80-100% with the relative standard deviations (RSDs) which were all less than 10.9%. The method was successfully applied to the tissue distribution research after oral administration of 6.0 g/kg APR extract to rat. The results revealed that the tissues distributions of four coumarins were in the liver, followed by the ovary, uterus, kidney, lung, heart, spleen, and muscle.

Simultaneous Determination of Columbianadin and Its Metabolite Columbianetin in Rat Plasma by LC-MS/MS: Application to Pharmacokinetics of Columbianadin after Oral Administration.

Columbianadin and its metabolite columbianetin exhibited the anti-inflammatory, analgesic, calcium channel blocking and antitumor activities. To compare the differences between pharmacokinetics of columbianadin and its metabolite columbianetin after oral administration of pure columbianadin and Angelicae Pubescentis Radix (APR) extract, a simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established and validated to simultaneously determine columbianadin and columbianetin in rat plasma. Two analytes and an internal standard (warfarin) were well separated and determined after liquid-liquid extraction with ethyl acetate. Ammonium acetate aqueous solution (1 mmol/L) and acetonitrile were used as the mobile phase and the flow rate was 0.3 mL/min. The lower limit of quantification (LLOQ) was 0.1 ng/mL for columbianetin and 0.5 ng/mL for columbianadin, respectively. There were significant differences between some pharmacokinetic parameters and bioavailability of columbianadin after oral administration of pure columbianadin and APR extract. The studies on comparative pharmacokinetics of columbianadin were of great use for facilitating the clinical application of columbianadin and were also highly meaningful for the potential development of APR.

Ultra high performance liquid chromatography with photodiode array detector and quadrupole time-of-flight tandem mass spectrometry coupled with discriminant analysis to evaluate Angelicae pubescentis radix from different regions.

A rapid and effective method was developed for the qualitative and quantitative analysis of the major chemical constituents in Angelicae pubescentis radix by ultra high performance liquid chromatography with photodiode array detection and quadrupole time-of-flight tandem mass spectrometry. The chromatographic separation was achieved on an ACQUITY UHPLC BEH C18 column (2.1 × 100 mm, 1.7 µm). Nine phenolic acids, 30 coumarins, bisabolangelone, and adenosine were identified by quadrupole time-of-flight tandem mass spectrometry. All calibration curves exhibited good linearity (r > 0.9996) within the linear ranges. The relative standard deviation calculated for intraday and interday precision, stability, and accuracy were <5%. The mean recovery ranged from 95.8 to 106%. The overall limits of detection and quantification were 0.025-0.160 and 0.100-0.560 µg/mL, respectively. Discriminant analysis was investigated as a method for evaluating the quality of the samples with 100% correction in their classification. The results demonstrated that the developed method could successfully be used to differentiate samples from different regions and could be a helpful tool for detection and confirmation of the quality of traditional Chinese medicines.

[Synthesis and characterization of dihydroeugenol acrylate].

Dihydroeugenol acrylate was synthesized by the reaction of acryloyl chloride (AC) with lignin mode compound dihydroeugenol (DH) in the presence of TEA and characterized by using FTIR, GC/MS, 1H-NMR and GPC. FTIR spectra showed that, after the esterification with acryloyl chloride, the intensity of stretching vibration peak of O-H (centered at 3 495 cm(-1)) of DH was disappeared. At the same time, a new peak appeared at 1 762 cm(-1) which was assigned to ester group. Additionally, the appearance of 1 631 and 981 cm(-1) were attributed to the carbon - carbon double bond confirmed the success in the synthesis of DH-AC. 1H-NMR spectra showed that, after the esterification with acryloyl chloride, the proton signal of O-H at 5.5 ppm was disappeared. Meanwhile, the appearance of three new proton signals at 6.0 ppm, 6.4 and 6.7 ppm, attributed to the vinylic protons, indicated that acryloyl chloride was successfully grafted onto DH. The results further confirmed the structures of the DH-AC. GC-MS results showed the DH-AC had a high purity of 98.63%. GPC results showed that dihydroeugenol acrylate could polymerize in the 1,4-dioxane using a thermal initiator of AIBN (2.0 Wt% of total monomers). The weight average molecular mass (Mw) of the homopolymer is 37 400 g x mol(-1), and the number average molecular mass is 23 400 g x mol(-1)' with a polydispersity index Mw/Mn of 1.60, indicating that the dihydroeugenol acrylate has high polymerization activity. This strategy provides a novel approach for extending the comprehensive utilization of lignin.

Body mass index and risk of gastric cancer: a meta-analysis.

OBJECTIVE: Overweight and obesity, indicated as increased body mass index, are associated with the risk of some cancers. We carried out a meta-analysis on published cohort and case-control studies to assess the strength of association between body mass index and gastric cancer. METHODS: Relevant studies were identified through PubMed, Web of Science and Medline electronic databases. Adjusted relative risks (odds ratios) with 95% confidence interval were used to assess the strength of association between body mass index and gastric cancer. RESULTS: Sixteen eligible studies were included in this meta-analysis. Overall, obesity (body mass index ≥ 30 kg/m(2)) was associated with an increased risk of gastric cancer (odds ratio = 1.13, 95% confidence interval = 1.03-1.24) compared with normal weight (body mass index = 18.5 to <25 kg/m(2)), while overweight (body mass index = 18.5 to <30 kg/m(2)) showed no association (odds ratio = 1.04, 95% confidence interval = 0.96-1.12). Specifically, a stratified analysis showed there were associations between obesity and the increased risk of gastric cancer for males (odds ratio = 1.27, 95% confidence interval = 1.09-1.48), non-Asians (odds ratio = 1.14, 95% confidence interval = 1.02-1.28) and both cohort studies (odds ratio = 1.10, 95% confidence interval = 1.00-1.22) and case-control studies (odds ratio = 1.29, 95% confidence interval = 1.03-1.60). Both overweight (odds ratio = 1.22, 95% confidence interval = 1.05-1.42) and obesity (odds ratio = 1.61, 95% confidence interval = 1.15-2.24) were associated with the increased risk of gastric cardia cancer. CONCLUSIONS: The results indicated that obesity was associated with the risk of gastric cancer, especially for males and among non-Asians. Both overweight and obesity were associated with the risk of gastric cardia cancer.

LC-MS/MS determination and pharmacokinetic study of columbianadin in rat plasma after intravenous administration of pure columbianadin.

BACKGROUND: Columbianadin, one of the active coumarins, is isolated from Radix Angelicae pubescentis which has been used as a traditional Chinese medicine for the treatment of rheumatic diseases for thousands of years. A fast and sensitive method is required for the determination of columbianadin for pharmacokinetic studies. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is a preeminent analytical tool for rapid biomedical analysis. RESULTS: A sensitive LC-MS/MS method has been validated to determine the concentration of columbianadin in rat plasma after intravenous administration of columbianadin (1, 2.5 and 5 mg kg(-1)). Liquid-liquid extraction was used to extract columbianadin from the rat plasma. Bergapten was selected as an internal standard (IS). The separations were performed on an Eclipse plus C18 column (4.6 × 100 mm, 1.8 µm) with ammonium acetate aqueous solution (1 mmol L(-1)) and acetonitrile as the mobile phase. The flow rate was set at 0.300 mL min(-1). Quantification was performed using multiple reaction monitoring (MRM) mode to monitor transitions of m/z 329.3 → 229.3 for columbianadin and m/z 217.2 → 202.2 for IS at positive ionization mode. The calibration curve was linear over the concentration range of 4-20000 ng mL(-1) with a correlation coefficient (r) of 0.996 or better. The precision of intra- and inter-batch assays ranged from 4.02 to 7.33% and accuracies determined at three concentrations ranged between 91.9% and 106%. The lower limit of quantification was about 4 ng mL(-1). CONCLUSION: The proposed LC-MS/MS method is simple, rapid and highly sensitive so that it could be used to evaluate pharmacokinetic properties of columbianadin in rat plasma after intravenous administration.

The pharmacokinetics and oral bioavailability studies of columbianetin in rats after oral and intravenous administration.

ETHNOPHARMACOLOGICAL RELEVANCE: The roots of Angelica pubescens Maxim. f. biserrata Shan et Yuan (RAP) has been used as Traditional Chinese medicine to treat rheumatic disease in China since ancient times, but its action mechanisms was not well understood. Columbianetin is one of the main active constituents isolated from RAP, which has been shown to have various biological activities, but the absorption characteristics and oral bioavailability dose proportionality of columbianetin in vivo were not studied. MATERIALS AND METHODS: Male Sprague Dawley rats (210-230 g) received either an intravenous (i.v. 5, 10 and 20 mg kg(-1)) or oral (5, 10 and 20 mg kg(-1)) dose of columbianetin. The levels of columbianetin in plasma were measured by a simple and sensitive reversed-phase high-performance liquid chromatography (HPLC) method. The simple liquid-liquid extraction with ethyl acetate was used for sample preparation. Osthole was selected as internal standard (IS). RESULTS: The chromatographic separation was accomplished on a C18 column at a flow rate of 1 mL min(-1), where water-methanol was used as mobile phase. The calibration curve of the method was linear in the concentration range of 0.05-2000 µg mL(-1). The intra and inter-day accuracy for columbianetin in rat plasma samples were within 8% and the variation was less than 8.3%. This method was suitable for the determination and pharmacokinetic study of columbianetin in rat plasma after both intravenous and oral administration. The results indicated that maximum plasma concentrations(Cmax) for the columbianetin (17-42 µg mL(-1)) were achieved at 0.3-0.5h post-oral dosing and the apparent volume of distribution (V/F) ranged from 0.38 to 0.44 L. Absolute bioavailability of columbianetin was assessed to be 81.13 ± 45.85, 81.09 ± 33.63 and 54.30 ± 23.19%, respectively. Terminal elimination half-life (T1/2) of the columbianetin after oral dosing was 60-90 min and were 2.5-3.3 fold longer than those observed for the i.v. dosing. CONCLUSIONS: The pharmacokinetic properties of columbianetin in rat after oral administration were characterized as rapid oral absorption, quick clearance and good absolute bioavailability. The bioavailability of columbianetin ranged from 54 to 81% for 5, 10 and 20 mg kg(-1) oral doses. The bioavailability of columbianetin is independent of the doses studied. Columbianetin showed dose proportionality over the dose range 5-20 mg kg(-1). The results clearly demonstrated that columbianetin was one of the material bases of RAP. Furthermore, an HPLC method was demonstrated in this study for the research of traditional Chinese medicine.

[Chemical structure of bioethanol lignin by low-temperature alkaline catalytic hydrothermal treatment].

In order to improve the reaction activity of bioethanol lignin, we investigated the activation of bioethanol lignin by a hydrothermal treatment method. Catalytic hydrothermal treatment of bioethanol lignin was performed at 180 degrees C for 3 h in the presence of alkaline solutions (NaOH, Na2 CO3, KOH and K2 CO3), the change in bioethanol lignin structures was studied comparatively by FTIR, 1H NMR,GPC and elemental analysis. FTIR spectra showed that after alkali hydrothermal treatment, the band at 1 375 cm(-1) attributed to the phenolic hydroxyl groups increased, and the band intensity at 1 116 cm(-1) attributed to the ether bond decreased. On the other hand, the band at 1 597 and 1 511 cm(-1) attributed to aromatic skeletal vibration remained almost unchanged. 1H NMR spectra showed that after alkali hydrothermal treatment, the number of aromatic methoxyl is increased, and based on the increment of the content of phenolic hydroxyl, the catalytic activity can be ranked as follows: KOH > NaOH > K2 CO3 > Na2 CO3. Especially for KOH, the increment of the content of phenolic hydroxyl was 170%, because the ion radius of potassium cation is bigger than sodium cation, so the potassium cations more easily formed cation adducts with lignin. GPC results showed that the molecular weight of alkali hydrothermal treatment lignin decreased and the molecular distribution got wider. Elemental analysis showed that hydrothermal treatment could break the interlinkage between lignin and protein, which can reduce the protein content and increase the purity of lignin, meanwhile, the content of O and H both decreased,while C fell, indicating that the bioethanol lignin had suffered a decarbonylation reaction. This is the most benefit of the lignin as a substitute for phenol.

[Renewable resource-based composites of thermoplastic acorn starch and polycaprolactone: preparation and FTIR spectrum analysis].

Acorn starch was used as the main material. Thermoplastic acorn starch (TPAS) and binary composites of thermoplastic acorn starch(TPAS)/Polycaprolactone (PCL) were prepared by hot-melt extrusion method. The effects of different plasticizers such as ethylene glycol, glycerol, monoethanolamine, iminobisetnanol and triethanolamine on molecular structure of samples were studied by FTIR and XRD analysis. In addition, the effects of different plasticizing system on molecular structure and mechanical properties of composites were also studied. The results showed that the addition of plasticizers changed the inter-molecular structure, and glycerol-based TPAS/PCL composites showed favorable mechanical properties.