Lactobacillus plantarum improves LPS-induced Caco2 cell line intestinal barrier damage via cyclic AMP-PKA signaling.

Lactobacillus plantarum (LP) has been shown to exhibit protective effects on intestinal barrier function in septic rats, although the regulatory mechanism has not been established. We determined whether LP imparts such protective effects in a lipopolysaccharide (LPS)-induced Caco2 cell monolayer model and whether cAMP-PKA signaling is the underlying mechanism of action. The cyclic adenosine monophosphate (cAMP) agonist, forskolin (FSK), and the protein kinase A (PKA) inhibitor, HT89, were used to study the protective effect of LP on the destruction of the tight junction (TJ) structure of cells treated with LPS and the corresponding changes in cAMP-PKA signaling. Our experimental results demonstrated that LP promoted the expression of TJ proteins between Caco2 cells after LPS treatment, and increased the electrical barrier detection (TEER) between Caco2 cells. Moreover, transmission electron microscopy (TEM) revealed that the TJ structural integrity of cells treated with LPS + LP was improved compared to cells treated with LPS alone. In addition, our findings were consistent between the FSK and LP intervention group, while HT89 inhibited LP influence. Taken together, our results indicate that LP has an improved protective effect on LPS-induced damage to the monolayer membrane barrier function of Caco2 cells and is regulated by the cAMP-PKA pathway.

Determining the invasiveness of pure ground-glass nodules using dual-energy spectral computed tomography.

BACKGROUND: The present work aimed to investigate the clinical application of using quantitative parameters generated in the unenhanced phase (UP) and venous phase (VP) in dual-energy spectral CT for differentiating the invasiveness of pure ground-glass nodule (pGGN). METHODS: Sixty-two patients with 66 pGGNs who underwent preoperative dual-energy spectral CT in UP and VP were evaluated retrospectively. Nodules were divided into three groups based on pathology: adenocarcinoma in situ (AIS, n=19), minimally invasive adenocarcinoma (MIA, n=22) (both in the preinvasive lesion group) and invasive adenocarcinoma (IA, n=25). The iodine concentration (IC) and water content (WC) in nodules were measured in material decomposition images. The nodule CT numbers and slopes(k) were measured on monochromatic images. All measurements, including the maximum diameter of nodules were statistically compared between the AIS-MIA group and IA group. RESULTS: There were significant differences of WC in VP between AIS-MIA group and IA group (P<0.05). The CT attenuation values of the 40-140 keV monochromatic images in UP and VP were significantly higher for the invasive nodules. Logistic regression analysis showed that the maximum nodule diameter [odd ratio (OR) =1.21, 95% CI: 1.050-1.400, P<0.01] and CT number in 130 keV images in venous phase (OR =1.03, 95% CI: 1.014-1.047, P<0.001) independently predicted histological invasiveness. CONCLUSIONS: The quantitative parameters in dual-energy spectral CT in the unenhanced phase and venous phase provide useful information in differentiating preinvasive lesion group from IA group of pGGN, especially the maximum nodule diameter and CT number in the 130 keV images in the venous phase.

Low-Frequency Sonophoresis of Chinese Medicine Formula Improves Efficacy of Malignant Pleural Effusion Treatment.

OBJECTIVE: To evaluate whether low-frequency ultrasound-facilitated transdermal delivery of a Chinese medicine (CM) formula could improve the efficacy of intrapleural administration of interleukin-2 (IL-2) in treatment of malignant pleural effusion (MPE). METHODS: A total of 110 eligible participants were randomized into the low-frequency sonophoresis (LFS) of CM (LSF/CM) group (55 cases) and the control group (55 cases) by simple randomization using a random number table. The control group was treated with an intrapleural administration of IL-2; and the LFS/CM group was treated with LFS of a CM gel formulation, combined with the same IL-2 injection as in the control group. The CM formula consisted of Semen Lepidii, Semen Sinapis, Ramulus Cinnamomi, Poriacocos, Herba Lycopi, and Radix Paeoniae Rubra. After 2-week treatment, the therapeutic outcome was determined by the change of the amount of MPE, which was evaluated by B-scan ultrasound and/or chest X-ray, and the change of quality of life (QOL) scores, which were evaluated by the Eastern Cooperative Oncology Group (ECOG) performance status. RESULTS: A significantly higher objective remission rate (ORR) was obtained with intrapleural IL-2 plus LFS/CM than IL-2 treatment alone (P=0.049). In addition, more patients in the LFS/CM group than in the control group had an improved QOL score (P=0.048), and no patients in the LFS/CM group had a reduced QOL. CONCLUSION: LFS of CM formulation could effectively alleviate MPE and improve the QOL of cancer patients.

Topical Delivery of Modified Da-Cheng-Qi Decoction () Using Low-Frequency Ultrasound Sonophoresis for Refractory Metastatic Malignant Bowel Obstruction: An Open-Label Single-Arm Clinical Trial.

OBJECTIVE: To evaluate the efficacy and safety of topical delivery of modified Da-Cheng- Qi Decoction (, MDCQD) by low-frequency ultrasound sonophoresis (LFUS) in patients with refractory metastatic malignant bowel obstruction (MBO) using an objective performance criteria (OPC) design. METHODS: Fifty patients with refractory metastatic MBO were enrolled in this open-label single-arm clinical trial. Alongside fasting, gastrointestinal decompression, glycerol enema, intravenous nutrition and antisecretory therapy, a 50 g dose of MDCQD (prepared as a hydrogel) was applied through topical delivery at the site of abodminal pain or Tianshu (S 25) using LFUS for 30 min, twice daily for 5 consecutive days. The overall outcome was the remission of intestinal obstruction, and improvement on abdominal pain, abdominal distention, nausea and vomiting scores. Indicators of safety evaluation included liver and renal function as well as blood coagulation indicators. RESULTS: Among 50 patients, 5 patients (10%) showed complete remission of intestinal obstruction and 21 patients (42%) showed improvement of intestinal obstruction. The overall remission rate of bowel obstruction was 52%. The results of the symptom score, based on the severity and frequency of the episode, are as follows: 26 patients (52%) showed improvment on symptom scores, 20 patients (40%) did not respond to treatment, and 4 patients (8%) discontinued treatment due to intolerance. No serious adverse effects or abnormal changes on liver and renal function or blood coagulation were observed. CONCLUSION: Topical delivery of MDCQD at 100 g/day using LFUS can improve the treatment response in patients with refractory metastatic MBO.

Image quality comparison of two adaptive statistical iterative reconstruction (ASiR, ASiR-V) algorithms and filtered back projection in routine liver CT.

OBJECTIVE: To compare image quality of two adaptive statistical iterative reconstruction (ASiR and ASiR-V) algorithms using objective and subjective metrics for routine liver CT, with the conventional filtered back projection (FBP) reconstructions as reference standards. METHODS: This institutional review board-approved study included 52 patients with clinically suspected hepatic metastases. Patients were divided equally into ASiR and ASiR-V groups with same scan parameters. Images were reconstructed with ASiR and ASiR-V from 0 (FBP) to 100% blending percentages at 10% interval in its respective group. Mean and standard deviation of CT numbers for liver parenchyma were recorded. Two experienced radiologists reviewed all images for image quality blindly and independently. Data were statistically analyzed. RESULTS: There was no difference in CT dose index between ASiR and ASiR-V groups. As the percentage of ASiR and ASiR-V increased from 10 to 100% , image noise reduced by 8.6 -57.9% and 8.9-81.6%, respectively, compared with FBP. There was substantial interobserver agreement in image quality assessment for ASiR and ASiR-V images. Compared with FBP reconstruction, subjective image quality scores of ASiR and ASiR-V improved significantly as percentage increased from 10 to 80% for ASiR (peaked at 50% with 32.2% noise reduction) and from 10 to 90% (peaked at 60% with 51.5% noise reduction) for ASiR-V. CONCLUSION: Both ASiR and ASiR-V improved the objective and subjective image quality for routine liver CT compared with FBP. ASiR-V provided further image quality improvement with higher acceptable percentage than ASiR, and ASiR-V60% had the highest image quality score. Advances in knowledge: (1) Both ASiR and ASiR-V significantly reduce image noise compared with conventional FBP reconstruction. (2) ASiR-V with 60 blending percentage provides the highest image quality score in routine liver CT.

A prospective evaluation of contrast and radiation dose and image quality in cardiac CT in children with complex congenital heart disease using low-concentration iodinated contrast agent and low tube voltage and current.

OBJECTIVE: To the assess image quality, contrast dose and radiation dose in cardiac CT in children with congenital heart disease (CHD) using low-concentration iodinated contrast agent and low tube voltage and current in comparison with standard dose protocol. METHODS: 110 patients with CHD were randomized to 1 of the 2 scan protocols: Group A (n = 45) with 120 mA tube current and contrast agent of 270 mgI/ml in concentration (Visipaque™; GE Healthcare Ireland, Co., Cork, UK); and Group B (n = 65) with the conventional 160 mA and 370 mgI/ml concentration contrast (Iopamiro®; Shanghai Bracco Sine Pharmaceutical Corp Ltd, Shanghai, China). Both groups used 80 kVp tube voltage and were reconstructed with 70% adaptive statistical iterative reconstruction algorithm. The CT value and noise in aortic arch were measured and the signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) were calculated. A five-point scale was used to subjectively evaluate image quality. Contrast and radiation dose were recorded. RESULTS: There was no difference in age and weight between the two groups (all p > 0.05). The iodine load and radiation dose in Group A were statistically lower (3976 ± 747 mgI vs 5763 ± 1018 mgI in iodine load and 0.60 ± 0.08 mSv vs 0.77 ± 0.10 mSv in effective dose; p < 0.001). However, image noise, CT value, CNR, SNR and subjective image quality for the two groups were similar (all p > 0.05), and with good agreement between the two observers. Comparing the surgery results, the diagnostic accuracy for extracardiac and intracardiac defects for Group A was 96% and 92%, respectively, while the corresponding numbers for Group B were 95% and 93%. CONCLUSION: Compared with the standard dose protocol, the use of low tube voltage (80 kVp), low tube current (120 mA) and low-concentration iodinated contrast agent (270 mgI/ml) enables a reduction of 30% in iodine load and 22% in radiation dose while maintaining compatible image quality and diagnostic accuracy. Advances in knowledge: The new cardiac CT scanning protocol can largely reduce the adverse effects of radiation and contrast media to children. Meanwhile, it also can be used effectively to examine complex CHD.

Safflower polysaccharide induces NSCLC cell apoptosis by inhibition of the Akt pathway.

Lung cancer is the leading cause of cancer death in the world. Safflower polysaccharide (SPS) has been used for the improvement of immunomodulatory activities and treatment of cancers. However, studies on the effect of SPS on the progression of lung cancer have rarely been reported. To study the antitumor effect of SPS on human lung cancer and its potential mechanism, non-small cell lung cancer cell lines (NSCLC), A549 and YTMLC-90 were treated with SPS at various concentrations ranging from 0.04 to 2.56 mg/ml and BALB/c nude tumor-bearing mice were injected intraperitoneally with SPS at concentrations ranging from 15 to 135 mg/kg. Results showed that SPS suppressed the proliferation of A549 and YTMLC-90 cells and induced apoptosis by increasing mRNA levels of bax and caspase-3, and inhibited tumor growth in vivo. SPS induced cell cycle arrest in the G2/M phase by decreasing the expression of cdc25B and cyclin B1. Moreover, SPS decreased the expression of Akt, p-Akt and PI3K. In mice, SPS injection enhanced immunomodulatory activities by increasing levels of TNF-α and IL-6 in tumor-bearing mice. Our findings suggest that SPS suppresses tumor growth by enhancing immunomodulatory activities and blocking the PI3K/Akt pathway. This study provides new insight into the anticancer mechanism of SPS.

Diagnostic accuracy of sub-mSv prospective ECG-triggering cardiac CT in young infant with complex congenital heart disease.

To explore the clinical value and evaluate the diagnostic accuracy of sub-mSv low-dose prospective ECG-triggering cardiac CT (CCT) in young infants with complex congenital heart disease (CHD). A total of 102 consecutive infant patients (53 boys and 49 girls with mean age of 2.9 ± 2.4 m and weight less than 5 kg) with complex CHD were prospectively enrolled. Scans were performed on a 64-slice high definition CT scanner with low dose prospective ECG-triggering mode and reconstructed with 80 % adaptive statistical iterative reconstruction algorithm. All studies were performed during free breathing with sedation. The subjective image quality was evaluated by 5-point grading scale and interobserver variability was calculated. The objective image noise (standard deviation, SD) and contrast to noise ratio (CNR) was calculated. The effective radiation dose from the prospective ECG-triggering mode was recorded and compared with the virtual conventional retrospective ECG-gating mode. The detection rate for the origin of coronary artery was calculated. All patients also underwent echocardiography before CCT examination. 81 patients had surgery and their preoperative CCT and echocardiography findings were compared with the surgical results and sensitivity, specificity, positive and negative predictive values and accuracy were calculated for separate cardiovascular anomalies. Heart rates were 70-161 beats per minute (bpm) with mean value of 129.19 ± 14.52 bpm. The effective dose of 0.53 ± 0.15 mSv in the prospective ECG-triggering cardiac CT was lower than the calculated value in a conventional retrospective ECG-gating mode (2.00 ± 0.35 mSv) (p < 0.001). The mean CNR and SD were 28.19 ± 13.00 and 15.75 ± 3.61HU, respectively. The image quality scores were 4.31 ± 0.36 and 4.29 ± 0.41 from reviewer 1 and 2 respectively with an excellent agreement between them (Kappa = 0.85). The detection rate for the origins of the left and right coronary arteries was 96 and 90 %, respectively. The detection rates of the origins of left coronary artery and right coronary artery in all cases were 96 % (78/81) and 90 % (73/81), respectively. Twenty cases of conotruncal anomalies and ALCAPA were validated surgically and the accuracy of cardiac CT diagnosis was 95 % (19/20). The overall deformity based sensitivity, specificity, positive predictive value and negative predictive value were 94.0.1, 99.9, 98.6, 99.5 % respectively, by CCT, and 88.2, 99.9, 97.8, 99.0 %, respectively, by echocardiography. Prospective ECG-triggering CCT with sub-mSv effective dose provides excellent imaging quality and high diagnostic accuracy for young infants with complex CHD.

Rapamycin, an mTOR inhibitor, induced apoptosis via independent mitochondrial and death receptor pathway in retinoblastoma Y79 cell.

Rapamycin is helpful in the treatment of certain cancers by inhibiting mTOR (mammalian target of rapamycin) pathway. Here, rapamycin mediated apoptosis were investigated in human retinoblastoma Y79 cells. The MTT assay showed that the IC50 value of rapamycin against Y79 cells was 0.136 ± 0.032 µmol/L. Flow cytometry analysis indicated that the percentage of apoptotic cells was increased from 2.16 ± 0.41% to 12.24 ± 3.10%, 20.16 ± 4.22%, and 31.32 ± 5.78% after 0.1, 0.2, and 0.4 µmol/L rapamycin or without rapamycin treatment for 48 hours. Flow cytometry analysis showed that rapamycin induced mitochondrial membrane potential (∆Ψm) collapse in Y79 cells in a concentration-dependent manner. Western blot assay showed that rapamycin led to release of cytochrome c from mitochondrial membranes to cytosol. Further Western blot assays showed that rapamycin induced activation of caspase-9 and caspase-8 and the cleavage of caspase-3. Rapamycin induced cleavages of caspase-3 and apoptosis was inhibited by both Z-LETD-FMK and Z-IETD-FMK treatment. Together, all these results illustrated that rapamycin induced apoptosis in human retinoblastoma Y79 cells involvement of both intrinsic and extrinsic pathways.

Rapamycin, a mTOR inhibitor, induced growth inhibition in retinoblastoma Y79 cell via down-regulation of Bmi-1.

Rapamycin is useful in the treatment of certain cancers by inhibiting mTOR(mammalian target of rapamycin) pathway. Here, anticancer activity and its acting mechanisms of rapamycin were investigated in human retinoblastoma Y79 cells. CCK-8 assay showed that the IC50 value of rapamycin against human retinoblastoma Y79 cells was 0.122±0.026 µmol/L. Flow cytometry analysis indicated that rapamycin induced G1 cell cycle arrest. Western blot assay demonstrated that the mTOR pathway in Y79 cells was blocked by rapamycin. Western blot and RT-PCR assay showed that Bmi-1 was downregulated in protein and mRNA level by rapamycin treatment. Further Western blot and RNA interference assays showed that rapamycin-mediated downregulation of Bmi-1 induced decreases of cyclin E1, which accounted for rapamycin-mediated G1 cell cycle arrest in human retinoblastoma cells. Together, all these results illustrated that rapamycin induced growth inhibition of human retinoblastoma cells, and inactive of mTOR pathway and downregulation of Bmi-1 was involved in its action mechanism.

[Preparation and evaluation of doxorubicin hydrochloride liposomes modified by poly(2-ethyl-2-oxazoline)-cholesteryl methyl carbonate].

In this study, the buffering capacity of amphiphilic pH-sensitivity copolymer poly(2-ethyl-2-oxazoline)-cholesteryl methyl carbonate (PEOZ-CHMC) was evaluated. The ammonium sulfate gradient method was used to prepare doxorubicin hydrochloride (DOX x HCl)-loaded liposomes (DOX-L), and then the post-insertion method was used to prepare PEOZ-CHMC and polyethylene glycol-distearoyl phosphatidyl ethanolamine (PEG-DSPE) modified DOX x HCl-loaded liposomes (PEOZ-DOX-L and PEG-DOX-L). The physico-chemical properties, in vitro drugs release behavior, cellular toxicity and intracellular delivery of liposomes were evaluated, separately. The results showed that PEOZ-CHMC has a satisfactory buffering capacity. The sephadex G-50 column centrifugation method and dynamic light scattering were used to determine the encapsulation efficiency (EE) and particle size of liposomes. The EE and particle size of DOX-L were (97.3 ± 1.4) % and 120 nm, respectively, and the addition of PEOZ-CHMC or PEG-DSPE had no influence on EE and particle size. The zeta potentials of three kinds of liposomes were negative. The release behavior of various DOX liposomes in vitro was investigated by dialysis method. In phosphate buffer solution (PBS) at pH 7.4, DOX x HCl was released from PEOZ-DOX-L in a sustained manner. While in PBS at pH 5.0, the release rate of DOX x HCl from PEOZ-DOX-L increased significantly, which suggested DOX x HCl was released from PEOZ-DOX-L in a pH-dependent manner. The intracellular delivery of liposomes was investigated by confocal laser scanning microscopy (CLSM). The CLSM images indicated that PEOZ-DOX-L showed efficient intracellular trafficking including endosomal escape and release DOX x HCl into nucleus, as well as the DOX-L and PEG-DOX-L had no this effect. The cytotoxicity of liposomes against MCF-7 cells was detected by using MTT assay. The results showed that antiproliferative effects of PEOZ-DOX-L enhanced with pH value decreased, whereas DOX-L and PEG-DOX-L did not have any significant difference in inhibitions at different pH conditions. Therefore, the problems of the inhibition of cellular uptake of liposomes and the failed endosomal escape of pH-sensitive liposomes by PEG chain can be overcome by the pH-sensitive liposomes constructed by PEOZ-CHMC.

Synthesis and biological evaluation of hydroxycinnamic acid hydrazide derivatives as inducer of caspase-3.

In order to generate compounds with superior antitumor activity and reduced toxicity, twelve new hydroxycinnamic acid hydrazide derivatives were synthesized and evaluated for their antiproliferative activities against two cancer cell lines (H1299 lung carcinoma cells and MCF-7 breast cancer cells), and compared to two normal counterparts (NL-20 lung epithelial cells and H184B5F5/M10 breast cells) by MTT method. The results demonstrated that some of these compounds possessed good antiproliferative activity against the two cancer cell lines. Among them, compound 2c was active against the growth of H1299 lung carcinoma cells with IC50 values of 1.50 µM, which was more active than the positive topotecan (IC50 = 4.18 µM). Simultaneously, it showed lower cytotoxic effects on normal NL-20 lung epithelial cells (IC50 > 10 µM). Mechanism studies indicated that it induced cell cycle arrest at G2/M phase followed by activation of caspase-3, and consequently caused the cell death. Further studies on the structure optimization are ongoing.

Brazilein, a compound isolated from Caesalpinia sappan Linn., induced growth inhibition in breast cancer cells via involvement of GSK-3ß/ß-Catenin/cyclin D1 pathway.

Caesalpinia sappan Linn. has long been used in traditional medicine in China. Here, the anticancer activity of brazilein, a compound isolated from C. sappan Linn. was investigated. MTT assay showed that the IC50 value of brazilein against human breast cancer MCF-7 cells was 7.23 ± 0.24 µmol/L. PI staining and flow cytometry analysis indicated that brazilein caused cell cycle arrest in G1 phase. Western blot and RT-PCR assay demonstrated that cyclin D1, a key factor of the G1 to S phase progression, was downregulated in a concentration-dependent manner by brazilein treatment. Further Western blot and RNA interference assay showed that brazilein treatment activated GSK-3ß and following reduced ß-Catenin protein, which accounted for the downregulation of cyclin D1 and blockage of cell cycle at G1 phase. Together, all these results illustrated that brazilein induced growth inhibition of breast cancer cells and downregulation of GSK-3ß/ß-Catenin pathway was involved in its action mechanism.

Differentiation of neoplastic from bland macroscopic portal vein thrombi using dual-energy spectral CT imaging: a pilot study.

OBJECTIVES: To assess the feasibility and value of dual-energy spectral computed tomography (DESCT) imaging for differentiating neoplastic from bland macroscopic portal vein (PV) thrombi. METHODS: Computed tomography (CT) images of 44 patients with macroscopic PV thrombus (bland group, n = 16; neoplastic group, n = 28) were reviewed. Iodine-based material decomposition images in the portal venous phase were reconstructed to compare the iodine indices between groups, including thrombus iodine density (I (T)), thrombus-aorta iodine density ratio (I (T)/I (A)), and thrombus-PV iodine density ratio (I (T)/I (P)). Differential diagnostic performances of DESCT were calculated in the subgroup of 21 patients with histopathological evidence (bland group, n = 12; neoplastic group, n = 9). RESULTS: The iodine indices of the neoplastic group were significantly higher than those in the bland group (P < 0.001). A threshold I (T) of 1.14 mg/mL, I (T)/I (A) of 0.17, and I (T)/I (P) of 0.17 in the portal venous phase yielded 100 %, 88.9 %, and 100 % sensitivity, and 91.7 %, 91.7 %, and 83.3 % specificity, respectively, in differentiating neoplastic from bland PV thrombi. CONCLUSIONS: DESCT imaging with quantification of thrombus iodine density in the portal venous phase appears to be a promising new method for distinguishing neoplastic from bland macroscopic PV thrombi. KEY POINTS: • Differentiating the nature of portal vein thrombus is of great clinical significance. • Iodine-based material decomposition imaging reflects iodine distribution after contrast media administration. • Dual-energy CT with iodine quantification can distinguish bland from neoplastic PV thrombi.

Dual energy spectral CT imaging of insulinoma-Value in preoperative diagnosis compared with conventional multi-detector CT.

OBJECTIVE: The aim of this study was to investigate the value of dual energy spectral CT (DEsCT) imaging in preoperative diagnosis of insulinomas in comparison with conventional multi-detector CT (MDCT). MATERIALS AND METHODS: Thirty-five patients were included in this study with 14 underwent the conventional dual-phase CT imaging (from March 2009 to January 2010) and 21 underwent the dual-phase DEsCT imaging (from February 2010 to May 2011). CT images were interpreted prospectively by two radiologists in consensus before operation. All the patients had diagnosis confirmed pathologically. The accuracy of preoperative diagnosis of insulinomas between DEsCT imaging and conventional MDCT, and between different kinds of images of DEsCT was compared. RESULTS: There were 39 confirmed lesions among the 35 patients (23 and 16 tumors in the spectral CT group and MDCT group, respectively). MDCT detected 11 of 16 tumors. DEsCT imaging detected 20 of 23 tumors separately with the monochromatic image or the iodine density image, and 22 of 23 tumors with the combination of the two kinds of images. The sensitivity for the preoperative diagnosis of insulinoma was 95.7% with the combination of monochromatic and iodine density images in DEsCT imaging, statistically higher than that with the conventional MDCT (68.8%) (p=0.033). CONCLUSION: Dual energy spectral CT imaging has higher sensitivity in preoperative diagnosis of insulinomas compared with conventional MDCT. The combination of monochromatic image and iodine density image can improve the diagnostic sensitivity of insulinomas.

[Effects of sodium butyrate on proliferation and differentiation of human gastric carcinoma cell line AGS].

BACKGROUND & OBJECTIVE: Sodium butyrate (NaBT) can inhibit proliferation and induce differentiation of various tumor cells. This study was to investigate effects of NaBT on the proliferation and differentiation of human gastric carcinoma cell line AGS and explore the possible mechanism. METHODS: AGS cells were treated with 0, 1.0, 2.0 and 4.0 mmol/L of NaBT. Cell proliferation was detected by MTT assay; cell morphology changes were observed under optical and transmission electron microscopy; cell cycle was detected by flow cytometry (FCM). The expression of cyclin-dependent kinase inhibitor p21 was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. RESULTS: After incubation with different concentrations of NaBT for 24 to 72 h, AGS cell proliferation was inhibited dramatically and the highest inhibition rate was 81.54%. The structure of AGS cells changed greatly. NaBT induced an increase of G0/G1 phase cells and a significant decrease of S phase cells accompanied by the changes in DNA ploidy. The expression of p21 was up-regulated at both mRNA and protein levels. NaBT exerted its effects in a dose-dependent manner. CONCLUSIONS: NaBT could induce G1 arrest and inhibit cell proliferation in AGS cells by up-regulating the expression of p21. This could reverse the malignant phenotype of AGS to some extents.

12-lipoxygenase induces apoptosis of human gastric cancer AGS cells via the ERK1/2 signal pathway.

12-Lipoxygenase (12-LOX) is over-expressed in a variety of human tumors, but its exact effect on the tumorogenesis of gastric cancer remains largely obscure. To investigate the effect of 12-LOX and its inhibitor baicalein on proliferation and apoptosis of human gastric cancer, AGS cells were separately treated with 12-hydroxyeicosatetraenoic acid (12-HETE, a metabolite of 12-LOX) and baicalein. MTT assay revealed that the absorbance of the 12-HETE-treated group was significantly (P < 0.01) higher than that of control group and that the absorbance of baicalein-treated group was significantly (P < 0.01) less than that of the control group, and that 48 h after treatment the apoptosis index of the baicalein-treated group was significantly (P < 0.01) higher than that of the untreated group and was significantly (P < 0.01) lower in the 12-HETE-treated group. Western blotting analysis was used to investigate the mechanism of these effects. The results revealed that the concentration of phosphorylated ERK in cells treated with 100 nmol L(-1) 12-HETE was significantly (P < 0.05) higher than in the untreated group and that the concentration of phosphorylated ERK1/2 in cells treated with 40 micromol L(-1) baicalein was significantly (P < 0.05) lower than in the untreated group. The expression level of bcl-2 was up-regulated and down-regulated after separate treatment with 12-HETE and baicalein, respectively, and both of these effects could be blocked by PD98059. Protein kinase C (PKC) activity was increased by treatment with 12-HETE and reduced by treatment with baicalein (P < 0.05). The PKC inhibitor BIM (bisindolymaleimide-I) blocked the phosphorylation of ERK1/2 and activation of PKC induced by 12-LOX. When pretreated with BIM, the concentration of phospho-ERK1/2 or bcl-2 in the BIM + 12-HETE-treated group was significantly (P < 0.05) lower than in that treated with 12-HETE only, and the concentration in the BIM + baicalein-treated group was significantly (P < 0.05) higher than in that treated with baicalein only. On the basis of these data we conclude that, via its metabolite 12-HETE, 12-LOX abolishes proliferation of AGS cells and protect cells from apoptosis by activating the ERK1/2 pathway and, eventually, enhances expression of bcl-2. Because PKC is also involved in the activation of ERK1/2 induced by 12-LOX, 12-LOX inhibitors would be potentially powerful anticancer agents for prevention and cure of human gastric cancer.

[Effect of the mutation of promoter region in Wilson disease ATP7B gene on the expression of reporter gene].

OBJECTIVE: To find out the relationship between mutation of ATP7B gene promoter region and pathogenesis of Wilson disease(WD). METHODS: Two of 48 WD patients presented C-->T base substitution mutations at the position -183. DNA sequences of the promoter region from normal and mutant samples were separated. The fragments containing the promoter region were cloned upstream of the luciferase. Luciferase activity was analyzed. RESULTS: The luciferase activity of reporter gene containing normal sequence of ATP7B gene promoter region did not show significant difference as compared with that of reporter gene containing mutant promoter(n=3, P > 0.05). CONCLUSION: No influence of C-->T base substitution mutations on the activity of promoter was observed in study. The results suggest that WD pathogenesis relates little to the mutations of the promoter region in Chinese.