[Relationship of DNMT3b expression and SEPT9 methylation in theprogression of colorectal carcinogenesis].

Objective: To investigate the relationship between the expression of DNA methyltransferase 3b (DNMT3b) and the methylation of SEPT9 gene, and their application prospects in the diagnosis and treatment of colorectal cancer. Methods: Seventy-five cases of colorectal cancer and adjacent tissues, 68 cases of colorectal high-grade internal neoplasia tissues (referred to as precancerous tissues) and high-grade internal adjacent neoplasia tissues (referred to as adjacent precancerous tissues) were collected. Pyrosequencing was used to detect the methylationlevel of SETP9. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to examine the mRNA expressionof SEPT9 and immunohistochemistry(IHC) was applied to detect the protein expressions of SETP9 and DNMT3b. Liposome-mediated method was used to transfect DNMT3b siRNA and negative control siRNA into HT-29 cells. Five groups including DNMT3b siRNA 15 nmol/L group, DNMT3b siRNA 30 nmol/L group, negative control siRNA 15 nmol/L group, negative control siRNA 30 nmol/L group and blank control group were set up. Pyrosequencing was applied to determine the methylation level of SEPT9 and mRNA expression of DNMT3b in each group. Results: The methylation rates of SEPT9 gene in colorectal cancer tissues, adjacent tissues, precancerous tissues and adjacent precancerous tissues were (76.8±6.5)%, (14.4±2.6)%, (34.6±5.0)% and (7.4±1.2)%, respectively, which was highest in colorectal cancer tissue (P<0.001). The relative expressions of SEPT9 mRNA were 0.18±0.03, 0.89±0.41, 0.69±0.41 and 1.01±0.21, respectively, which was lowest in colorectal cancer tissue (P<0.001), while there were no statistically significant differences in adjacent tissues, precancerous tissues and adjacent precancerous tissues (P>0.05). The positive rates of SEPT9 protein expression were 12.0% (9/75), 53.3% (40/75), 55.1% (38/69) and 62.3% (43/69), which was lowest in the colorectal cancer tissue (P<0.001), while there were no statistically significant differences in the adjacent group, precancerous group and adjacent precancerous group (P>0.016 7). The positive rates of DNMT3b protein expression were 56.3% (45/75), 26.7% (20/75), 46.4% (32/69) and 33.3% (23/69), respectively, which was highest in colorectal cancer tissue (P<0.001), while without statistically significant difference from the precancerous tissue (P>0.016 7). Experiments in vitro showed that DNMT3b mRNA expression was lowest in DNMT3b siRNA 30 nmol/L group among five groups and was statistically different from other groups (all P<0.05). Meanwhile, the methylationrate of SEPT9 gene was lowest in this group, but without statistically significant difference from the DNMT3b siRNA 15 nmol/L group (P>0.05). Conclusions: The expression of DNMT3b is significantly correlated with the methylation level of SEPT9 gene in different stages of colorectal cancer. The high expression of DNMT3b may be an important molecular event before SEPT9 gene methylation and it may have an important potential application value in the diagnosis and treatment of early colorectal cancer.

Diffusion-limited PBPK model for predicting pulmonary pharmacokinetics of florfenicol in pig.

For most bacterial lung infections, the concentration of unbound antimicrobial agent in lung interstitial fluid has been thought to be responsible for antimicrobial efficacy. In this study, a diffusion-limited physiologically based pharmacokinetic (PBPK) model was developed to predict the pulmonary pharmacokinetics of florfenicol (FF) in pigs. The model included separate compartments corresponding to blood, diffusion-limited lung, flow-limited muscle, liver, and kidney and an extra compartment representing the remaining carcass. The absorption rate constant and renal and hepatic clearance of FF were determined in vivo. Other parameters were taken from the literature or optimized based on existing pharmacokinetic data. All mathematical operations during the development of the model were performed using acslXtreme version 3.0.2.1 (Aegis Technologies Group, Inc., Huntsville, AL, USA). The model accurately predicted the concentration-time courses of FF in lung interstitial fluid, serum, and plasma following different dosing schedules, except at the dose of 15 mg/kg. When compared with the tissue residue data, the model generally underestimated the FF concentration at the injection site, whereas it gave good predictions of FF concentrations in lung, liver, and kidney at early time points. The model predictions provide a scientific basis for the dosage regimen design of FF.

Comparison of catalytical activity and stereoselectivity between the recombinant human cytochrome P450 2D6.1 and 2D6.10.

Polymorphisms of the cytochrome P450 2D6 (CYP2D6) gene play a major role in pharmacokinetic variability in human, while CYP2D6*10 is an important subtype in Asian people. In this study, the co-expression enzyme of human recombinant CYPOR, CYPb5 and CYP2D6.1 or CYP2D6.10 with the Bac-to-Bac system in baculovirus-infected insect cells was used to study the catalytical activity to imipramine metabolism and stereoselective metabolism of propranolol. The metabolites of imipramine were identified of hydroxyl imipramine and desipramine by LC-MS/MS. There are some differences between CYP2D6.1 and CYP2D6.10 activity. The kinetics parameters K(m), V(max), and CL(int) are 11.77 +/- 0.91 micromol/L, 0.4235 +/- 0.05 nmol/nmol CYP2D6.1/min and 3.60 x 10(-5) ml/min/nmol CYP2D6.1 (n = 3) for CYP2D6.1, respectively, and 9.05 +/- 0.87 micromol/L, 0.42 +/- 0.03 nmol/nmol CYP2D6.10/min, and 4.60 x 10(-5) ml/min/nmol CYP2D6.10 (n = 3) for CYP2D6.10. For propranolol, two metabolites were identified to be hydroxyl and N-desisopropylation propranolol by LC-MS/MS. When the substrate concentration was 0.20 micromol/L, CYP2D6.1 and CYP2D6.10 exhibited significant stereoseletivity. Furthermore, enantioselective formation has been detected. Both of CYP2D6.1 and CYP2D6.10 produced more hydroxyl propranolol from the R-(+)-isomer than from the S-(-)-isomer while there was no obvious difference for N-desisopropylation propranolol production between R-(+)- and S-(-)- isomer. In summary, there is a somewhat different catalytical activity and stereoselectivity between the human recombinant CYP2D6.1 and CYP2D6.10. The data we got will be helpful in preclinical research and clinical use of CYP2D6 substrates.