Combined Therapy with Rheum tanguticum Polysaccharide and Low-dose 5-ASA Ameliorates TNBS-Induced Colitis in Rats by Suppression of NF-κB.

The most common conventional therapy for inflammatory bowel disease in clinical practice involves the use of nonsteroidal anti-inflammatory drugs, such as 5-amino salicylic acid. However, a high dose of 5-amino salicylic acid may bring about severe side effects. Chinese people have used Rheum tanguticum as a folk remedy for gastrointestinal disease for two thousand years. Our group has isolated R. tanguticum polysaccharide 1 from R. tanguticum and verified that it can attenuate 2,4,6-trinitrobenzene sulfonic acid-induced colitis in murines/rats. The present study aims to evaluate whether the addition of R. tanguticum polysaccharide 1 can improve efficacy and limit subsequent side effects of conventional treatment (5-amino salicylic acid) in rats with 2,4,6-trinitrobenzene sulfonic acid-induced colitis. Sixty Sprague-Dawley male rats were randomized into five groups and treated with (1) saline (saline, 0.2 mL/day × 5, p. o.), (2) 2,4,6-trinitrobenzene sulfonic acid alone (saline, 0.2 mL/day × 5, p. o.), (3) 2,4,6-trinitrobenzene sulfonic acid + 5-amino salicylic acid (5-amino salicylic acid, 75 mg/kg/day × 5, p.o), (4) 2,4,6-trinitrobenzene sulfonic acid + R. tanguticum polysaccharide 1 (R. tanguticum polysaccharide 1, 200 mg/kg/day × 5, p. o.), and (5) 2,4,6-trinitrobenzene sulfonic acid + 5-amino salicylic acid + R. tanguticum polysaccharide 1 (5-amino salicylic acid, 25 mg/kg/day × 5, p.o; R. tanguticum polysaccharide 1, 200 mg/kg/day × 5, p. o.). All the rats were sacrificed on the 6th day after treatment using an overdose of anesthesia. A histological assessment was performed using semiquantitative scores; nuclear factor-kappa B and tumor necrosis factor-α were measured with Western blot, cyclooxygenase 1 and cyclooxygenase 2 protein expressions were investigated by RT-polymerase chain reaction, and prostoglandin E2 and inducible nitric oxide synthase productions were investigated by ELISA. The extent and severity of histological signs were attenuated significantly in the 2,4,6-trinitrobenzene sulfonic acid + 5-amino salicylic acid + R. tanguticum polysaccharide 1 group. Treatment with R. tanguticum polysaccharide 1 plus 5-amino salicylic acid markedly decreased nuclear factor-kappa Bp65 and tumor necrosis factor-α protein expressions. R. tanguticum polysaccharide 1 and 5-amino salicylic acid had no effect on cyclooxygenase 1 protein expression, but inhibited the overexpression of the cyclooxygenase 2 protein. After treatment with 5-amino salicylic acid and R. tanguticum polysaccharide 1, the prostoglandin E2 level increased significantly and the inducible nitric oxide synthase level decreased considerably in the 2,4,6-trinitrobenzene sulfonic acid + 5-amino salicylic acid + R. tanguticum polysaccharide 1 group compared with the 2,4,6-trinitrobenzene sulfonic acid alone group. These results demonstrate that combined therapy with R. tanguticum polysaccharide 1 and low-dose 5-amino salicylic acid had more favorable effects on 2,4,6-trinitrobenzene sulfonic acid-induced colitis in rats, and its effects may be associated with inhibiting nuclear factor-kappa Bp65 protein expression and tumor necrosis factor-α production, resulting in a decrease of cyclooxygenase 2 and inducible nitric oxide synthase protein expressions.

Antitumor activity of a sulfated polysaccharide from Enteromorpha intestinalis targeted against hepatoma through mitochondrial pathway.

A sulfated polysaccharide (EI-SP), extracted from Enteromorpha intestinalis that is a kind of algae, is found to have anticancer activity. This study was designed to investigate the anti-tumor effect of EI-SP on human hepatoma HepG2 cell line and its possible mechanisms. An MTT assay showed that EI-SP could specifically inhibit the growth of human hepatoma HepG2 cells in a dose-dependent manner. Analysis by flow cytometry indicated that the apoptosis of tumor cells increased after treatment with EI-SP in range of 100-400 µg/ml. Furthermore, Western blot analysis showed that EI-SP treatment led to decreased protein expression of Bcl-2 and an increase in Bax, cleaved caspase-3, cleaved caspase-9 and cleaved poly(ADP-ribose) polymerase (PARP). Moreover, it was found that EI-SP caused a loss of mitochondrial membrane potential (Δψ m) and the release of cytochrome c to the cytosol. Collectively, our results showed that the EI-SP induces apoptosis in HepG2 cells involving a caspases-mediated mitochondrial signalling pathway.

Optical enhancement mode 2 improves the detection rate of gastric neoplastic lesion in high-risk populations: A multicenter randomized controlled clinical study.

OBJECTIVES: Detection of early neoplastic lesions is crucial for improving the survival rates of patients with gastric cancer. Optical enhancement mode 2 is a new image-enhanced endoscopic technique that offers bright images and can improve the visibility of neoplastic lesions. This study aimed to compare the detection of neoplastic lesions with optical enhancement mode 2 and white-light imaging (WLI) in a high-risk population. METHODS: In this prospective multicenter randomized controlled trial, patients were randomly assigned to optical enhancement mode 2 or WLI groups. Detection of suspicious neoplastic lesions during the examinations was recorded, and pathological diagnoses served as the gold standard. RESULTS: A total of 1211 and 1219 individuals were included in the optical enhancement mode 2 and WLI groups, respectively. The detection rate of neoplastic lesions was significantly higher in the optical enhancement mode 2 group (5.1% vs. 1.9%; risk ratio, 2.656 [95% confidence interval, 1.630-4.330]; p < 0.001). The detection rate of neoplastic lesions with an atrophic gastritis background was significantly higher in the optical enhancement mode 2 group (8.6% vs. 2.6%, p < 0.001). The optical enhancement mode 2 group also had a higher detection rate among endoscopists with different experiences. CONCLUSIONS: Optical enhancement mode 2 was more effective than WLI for detecting neoplastic lesions in the stomach, and can serve as a new method for screening early gastric cancer in clinical practice. CLINICAL REGISTRY: United States National Library of Medicine (https://www. CLINICALTRIALS: gov), ID: NCT040720521.

The lncRNA MIR99AHG directs alternative splicing of SMARCA1 by PTBP1 to enable invadopodia formation in colorectal cancer cells.

Alternative splicing regulates gene expression and functional diversity and is often dysregulated in human cancers. Here, we discovered that the long noncoding RNA (lncRNA) MIR99AHG regulated alternative splicing to alter the activity of a chromatin remodeler and promote metastatic behaviors in colorectal cancer (CRC). MIR99AHG was abundant in invasive CRC cells and metastatic tumors from patients and promoted motility and invasion in cultured CRC cells. MIR99AHG bound to and stabilized the RNA splicing factor PTBP1, and this complex increased cassette exon inclusion in the mRNA encoding the chromatin remodeling gene SMARCA1. Specifically, MIR99AHG altered the nature of PTBP1 binding to the splice sites on intron 12 of SMARCA1 pre-mRNA, thereby triggering a splicing switch from skipping to including exon 13 to produce the long isoform, SMARCA1-L. SMARCA1, but not SMARCA1-L, suppressed invadopodia formation, cell migration, and invasion. Analysis of CRC samples revealed that the abundance of MIR99AHG transcript positively correlated with that of SMARCA1-L mRNA and PTBP1 protein and with poor prognosis in patients with CRC. Furthermore, TGF-ß1 secretion from cancer-associated fibroblasts increased MIR99AHG expression in CRC cells. Our findings identify an lncRNA that is induced by cues from the tumor microenvironment and that interacts with PTBP1 to regulate alternative splicing, potentially providing a therapeutic target and predictive biomarker for metastatic CRC.

Curcumin attenuates insulin resistance in hepatocytes by inducing Nrf2 nuclear translocation.

BACKGROUND/AIMS: NF-E2-Related Factor-2 (Nrf2) is a transcription factor that plays a crucial role in the cellular protection against oxidative stress. Curcumin has been reported to induce Nrf2 nuclear translocation and upregulate the expression of numerous reactive oxygen species (ROS) detoxifying and antioxidant genes in hepatocytes. This study was designed to investigate whether curcumin-induced Nrf2 nuclear translocation could reduce ROS-mediated insulin resistance in cultured LO2 hepatocytes. METHODOLOGY: Human LO2 hepatocytes were incubated with curcumine and glucose oxidase (GO) in the presence/absence of wortmannin (a phosphatidyinositol 3-kinase (PI3K) inhibitor), oxidative stress, cellular damage, Nrf2 nuclear translocation and insulin resistance were measured. RESULTS: GO exposure significantly increased intracellular ROS, glutathione (GSH) depletion, malondialdehyde (MDA) formation, and increased activities of cellular lactate dehydrogenase (LDH) and aspartate amino transferase (AST), as well as causing insulin resistance. Curcumin pretreatment significantly attenuated these disturbances in intracellular ROS, liver enzyme activity and significantly antagonized the lipid peroxidation, GSH depletion and insulin resistance induced by GO in LO2 hepatocytes. These effects paralleled Nrf2 nuclear translocation induced by curcumin. Wortmannin partially blocked curcumin-induced Nrf2 nuclear translocation. In addition, wortmannin prevented curcumin-induced improvements in intracellular ROS, MDA formation, GSH depletion, liver enzyme activity and insulin resistance in cultured LO2 hepatocytes. CONCLUSIONS: These findings suggest that curcumin could reduce ROS-mediated insulin resistance in hepatocytes, at least in part through nuclear translocation of Nrf2.

The roles of PDGFRα signaling in the postnatal development and functional maintenance of the SMC-ICC-PDGFRα+ cell (SIP) syncytium in the colon.

BACKGROUND: The SIP syncytium in the gut consists of smooth muscle cells, interstitial cells of Cajal, and PDGFRα+ cells. We studied the fate of SIP cells after blocking PDGFRα receptor to explore the roles of PDGFRα signaling in the postnatal development and functional maintenance of the SIP syncytium. METHODS: Crenolanib was administered to mice from P0, P10, or P50. The morphological changes in SIP cells were examined by immunofluorescence. Protein expression in SIP cells was detected by Western blotting. Moreover, colonic transit was analyzed by testing the colonic bead expulsion time. KEY RESULTS: A dose of 5 mg(kg•day)-1 crenolanib administered for 10 days beginning on P0 apparently hindered the development of PDGFRα+ cells in the colonic longitudinal muscularis and myenteric plexus without influencing their proliferative activity and apoptosis, but this result was not seen in the colonic circular muscularis. SMCs were also inhibited by crenolanib. A dose of 7.5 mg(kg•day)-1 crenolanib administered for 15 days beginning on P0 caused reductions in both PDGFRα+ cells and ICC in the longitudinal muscularis, myenteric plexus, and circular muscularis. However, when crenolanib was administered at a dose of 5 mg(kg•day)-1 beginning on P10 or P50, it only noticeably decreased the number of PDGFRα+ cells in the colonic longitudinal muscularis. Crenolanib also caused PDGFRα+ cells to transdifferentiate into SMC in adult mice. Colonic transit was delayed after administration of crenolanib. CONCLUSIONS & INFERENCES: Therefore, PDGFRα signaling is essential for the development and functional maintenance of the SIP cells, especially PDGFRα+ cells.

Identification and distribution of thioredoxin-like 2 as the antigen for the monoclonal antibody MC3 specific to colorectal cancer.

MC3 is a colorectal cancer (CRC)-specific mAb previously prepared in our laboratory that can detect CRC with high sensitivity and specificity. However, the target antigen for MC3 had not been identified due to technological limitations. In the present study, immunocytochemistry and immunohistochemistry revealed the expression patterns of MC3 antigen (MC3-Ag) in colon cancer cell lines and CRC tissues. Western blotting analysis showed that the MC3 antibody reproducibly recognized two approximately 30 kDa proteins in the total cell lysates of human colon carcinoma cell lines SW480 and HT-29. Using a proteomic approach, we identified two MC3 immunoreactive spots as two isoforms of thioredoxin-like 2 (Txl-2) protein. Further paired immunostaining showed that Txl-2 had the same expression profile as probed by the MC3 antibody. Western blotting also showed that both antibodies could detect the same two bands, further verifying that Txl-2 is the antigen of MC3 antibody. Additionally, tissue arrays revealed the expression patterns of Txl-2 in various normal and cancer tissues. Further analysis showed that Txl-2 mRNA was elevated in 18 cases of CRC tissues compared to paracancerous tissues and adjacent normal tissues.

Expression of 15-PGDH is downregulated by COX-2 in gastric cancer.

To explore the proteins regulated by cyclooxygenase-2 (COX-2) in gastric cancer, the expression plasmid of COX-2siRNA was constructed and transfected into gastric cancer cell line SGC7901. Then, two-dimensional electrophoresis and the PDQuest software analysis were applied to discover the differentially expressed proteins. The differential protein spots were analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry. Fourteen differentially expressed proteins between the two cell lines were identified. 15-Hydroxyprostaglandin dehydrogenase [NAD(+)] (15-PGDH), a key enzyme in prostaglandin degradation, was identified as an upregulated protein in SGC7901 cells transfected with the COX-2siRNA plasmid. To further explore whether the 15-PGDH is regulated by COX-2, western blotting and immunocytochemical assay were performed to detect the expression of 15-PGDH in different cell lines with different expression level of COX-2. The results showed that the expression of 15-PGDH was upregulated (128.57%) as COX-2 was suppressed by small interfering RNA and downregulated (51.72%) as COX-2 was enhanced by COX-2 cDNA transfection in gastric cancer cells. In tissue specimens with gastric cancer, there was a decreased expression of 15-PGDH and an increased expression of COX-2 simultaneously. A significantly negative correlation of 15-PGDH expression was found to COX-2 level, tumor differentiation, tumor, lymph node, metastasis (TNM) staging and lymph node metastasis of gastric cancer. All the results suggest that 15-PGDH is downregulated by COX-2 in human gastric cancer and may contribute to the carcinogenesis and development of human gastric cancer in combination with COX-2.

Identification of triosephosphate isomerase as an anti-drug resistance agent in human gastric cancer cells using functional proteomic analysis.

AIMS: Proteomic study was used to explore new multidrug resistance (MDR)-related proteins and clarify novel mechanism of MDR in gastric cancer. METHODS: Two-dimensional gel electrophoresis and the PDQuest software analysis were applied to compare the differential expression of MDR-related proteins in gastric cancer SGC7901 cells and drug-resistant SGC7901 cells (SGC7901/VCR) induced by vincristine sulfate (VCR). The differential protein dots were excised and further analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis (MALDI-TOF-MS). RESULTS: Nine differential expression proteins between the two cell lines were successfully identified by MALDI-TOF-MS. Triosephosphate isomerase (TPI), a glycolytic pathway enzyme, was identified as a downregulated protein in SGC7901/VCR cells. Further, Western blot analysis and semiquantitative RT-PCR confirmed its decreased expression in SGC7901/VCR cells. Sense vector pcDNA3.1-TPI was constructed and transfected into SGC7901/VCR. The sensitivity of TPI-SGC7901/VCR cells to adriamycin (ADR), VCR, 5-fluorouracil and cis-dichlorodiamine platinum, as well as the accumulation and retention to ADR, were significantly increased when compared to their control cell lines. CONCLUSIONS: These results provide new MDR-related protein candidates, which are differentially expressed in the MDR cell line and its parental cell line including TPI, which may participate in the VCR-mediated MDR in human gastric cancer. Upregulation of TPI expression could partially reverse multidrug-resistant phenotype of SGC7901/VCR, which suggests that TPI may be an anti-drug resistance agent in gastric cancer and the candidate target to develop novel therapeutics for better treatment of gastric cancer.

Effects of essential oil from Croton tiglium L. on intestinal transit in mice.

AIM OF THE STUDY: Croton tiglium (Croton tiglium L., Euphorbiaceae) is widely used as a herb for treatment of gastrointestinal disturbances. Previous studies established its purgative and inflammational properties. The present study aimed to investigate the effects of Croton tiglium oil (CO) on intestinal transit in mice. MATERIALS AND METHODS: Gastrointestinal transit in mice and contractile characteristics of isolated intestinal strips from mice were evaluated. Intestinal inflammation was confirmed by histological examination. RESULTS: Low dose of CO increased the gastrointestinal transit of charcoal and barium meal as well as the production of fecal pellets in mice. In contrast, high dose exerted inhibitory effects. For normal colonic circular strips, both high and low dose of CO inhibited the contractile frequency. Low doses (0-20 microg/ml) of CO enhanced the phasic contractions, while high doses (>40 microg/ml) reduced them. Colonic longitudinal strips in CO-treated mice were less sensitive to electrical field stimulation than those in control mice. The contraction of colonic longitudinal, colonic and jejunal circular strips in CO-treated mice was more sensitive to atropine than that in control mice. CONCLUSIONS: CO might modulate gastrointestinal motility and induce intestinal inflammation related to immunological milieu and motor activity. Our findings may highlight the ethno-medical uses of Croton tiglium on intestinal disorders.

Effect of explosive noise on gastrointestinal transit and plasma levels of polypeptide hormones.

AIM: To investigate the effect of firing noise on gastrointestinal transit and probe its mechanism by measuring the levels of plasma polypeptide hormones. METHODS: A total of 64 SD rats were randomly divided into a control group and three stimulating groups. Firing noise of different intensity by sub-machine guns was used as inflicting factor. The effect of firing noise on liquid substance gastrointestinal transit and solid substance gastrointestinal transit was observed by measuring the ratio of carbon powder suspension transmitting and barium sticks transmitting respectively. Plasma levels of polypeptide hormones were measured by radio-immunoassay. RESULTS: The noise accelerated gastrointestinal transit of solid food by more than 80 db;and accelerated gastrointestinal transit of liquid food significantly by more than 120 db. Meantime, plasma levels of plasma motilin (MTL)(157.47+/-16.08; 151.90+/-17.08), somatostatin (SS)(513.97+/-88.77; 458.25+/-104.30), substance P (SP)(115.52+/-20.70; 110.28+/-19.96) and vasoactive intestinal peptide (VIP) (214.21+/-63.17; 251.76+/-97.24) remarkably changed also. CONCLUSION: Within a certain intensity range, the firing noise changes the levels of rat plasma gastrointestinal hormones, but the gastrointestinal transit is still normal. Beyond the range, the noise induces plasma hormone levels disturbance and gastrointestinal transit disorder.

[Intraoperative and postoperative intra-peritoneal administration of peptide PIII inhibits peritoneal metastasis of gastric cancer].

OBJECTIVE: To evaluate the preventive potential of intraoperative or early postoperative local administration of peptide PIII into the abdominal cavity against peritoneal carcinomatosis. METHODS: Human gastric cancer cells of the line GC9811 were inoculated subcutaneously into nude mice to cause subcutaneous carcinoma. The 8th generation cancer was taken out to isolate the cancer cells to be inoculated into the serous membrane at the greater curvature of the stomach. Forty-eight nude BALB/C-nu/nu mice underwent implantation of the 8th generation cancer cells into the serous membrane at the greater curvature of the stomach via minilaparotomy so as to establish models of peritoneal carcinoma The 48 mice were randomly divided into 3 equal groups: Group 1 (control group); Group 2, undergoing intraperitoneal perfusion of 100 microg of peptide PIII, and post-operative intraperitoneal perfusion of 2 mg/kg peptide PIII twice; and Group 3, undergoing post-operative intraperitoneal perfusion of 100 microg/kg peptide PIII twice. Six mice in every group were sacrificed on the day 23 to undergo pathological examination. The other mice in every group were used to evaluate the survival rate. The tumor-bearing mice showing manifestations of failure were killed to undergo pathology. RESULTS: The weight of individual tumor was not significantly different among the 3 groups. The number of peritoneal metastatic nodules of Group 2 was (9.2 +/- 1.3), significantly less than those of Group 1 and 3 [(126.3 +/- 9.6) and (64.2 +/- 8.3) respectively, both P < 0.01]. The number of metastatic nodules > 2 mm of Group 2 was 1.6 +/- 0.2, significantly less then those of Groups 1 and 3 [(51.2 +/- 3.6) and (21.7 +/- 4.9) respectively, both P < 0.01]. The survival rate of Group 2 was significantly longer than those of Groups 1 and 3 (both P < 0.01). CONCLUSION: Peptide PIII does not significantly inhibit the growth of GC, but significantly reduce the peritoneal metastasis of GC. An ideal targeting chemotherapy, intra-operative application of peptide PIII into the abdominal cavity plus intraperitoneal perfusion is an attractive and promising strategy to prevent peritoneal metastasis of GC.

Deletion of Nrf2 leads to hepatic insulin resistance via the activation of NF-κB in mice fed a high-fat diet.

Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome. Insulin resistance (IR) is important in the development and progression of NAFLD. Nuclear erythroid 2­related factor 2 (Nrf2) has previously been reported to be a novel regulator in NAFLD. The present study determined that Nrf2 knockdown accelerated the onset of obesity and non­alcoholic steatohepatitis (NASH), via the induction of hepatic IR in mice fed a high­fat diet (HFD), which was confirmed by an increase in total and hepatic weight in Nrf2­null­HFD mice, in addition to marked structural disorder in liver tissues from the Nrf2­null­HFD group analyzed by histopathological examination. Subsequently, it was demonstrated that hepatic IR in Nrf2­null­HFD mice was influenced by oxidative stress; this was confirmed by an increase in malondialdehyde levels and a decrease in glutathione levels. In addition, it was determined that the induction of hepatic IR by Nrf2 knockdown in HFD-treated mice was regulated by activation of the nuclear factor­κB (NF­κB) signaling pathway, as detected by an increase in the expression levels of nuclear NF­κB, and its downstream effectors interleukin­6 and tumor necrosis factor­α. The present study provides insight into the function of Nrf2 in NAFLD, indicating that Nrf2 deletion may lead to hepatic IR by activation of NF­κB, which is often associated with oxidative stress. Therefore, activation of Nrf2 may limit disease progression and act as a therapeutic approach for the treatment of NASH.

CacyBP/SIP nuclear translocation regulates p27Kip1 stability in gastric cancer cells.

AIM: To investigate the mechanism of calcyclin binding protein/Siah-1 interacting protein (CacyBP/SIP) nuclear translocation in promoting the proliferation of gastric cancer (GC) cells. METHODS: The effect of CacyBP/SIP nuclear translocation on cell cycle was investigated by cell cycle analysis. Western blot analysis was used to assess the change in expression of cell cycle regulatory proteins and proteasome-mediated degradation of p27Kip1. Co-immunoprecipitation (co-IP) analysis was performed to examine the binding of CacyBP/SIP with Skp1. A CacyBP/SIP truncation mutant which lacked the Skp1 binding site was constructed and fused to a fluorescent protein. Subsequently, the effect on Skp1 binding with the fusion protein was examined by co-IP, while localization of fluorescent fusion protein observed by confocal laser microscopy, and change in p27Kip1 protein expression assessed by Western blot analysis. RESULTS: CacyBP/SIP nuclear translocation induced by gastrin promoted progression of GC cells from G1 phase. However, while CacyBP/SIP nuclear translocation was inhibited using siRNA to suppress CacyBP/SIP expression, cell cycle was clearly inhibited. CacyBP/SIP nuclear translocation significantly decreased the level of cell cycle inhibitor p27Kip1, increased Cyclin E protein expression whereas the levels of Skp1, Skp2, and CDK2 were not affected. Upon inhibition of CacyBP/SIP nuclear translocation, there were no changes in protein levels of p27Kip1 and Cyclin E, while p27Kip1 decrease could be prevented by the proteasome inhibitor MG132. Moreover, CacyBP/SIP was found to bind to Skp1 by immunoprecipitation, an event that was abolished by mutant CacyBP/SIP, which also failed to stimulate p27Kip1 degradation, even though the mutant could still translocate into the nucleus. CONCLUSION: CacyBP/SIP nuclear translocation contributes to the proliferation of GC cells, and CacyBP/SIP exerts this effect, at least in part, by stimulating ubiquitin-mediated degradation of p27Kip1.

Curcumin upregulates Nrf2 nuclear translocation and protects rat hepatic stellate cells against oxidative stress.

The present study aimed to investigate the protective role of curcumin against oxidative stress in rat hepatic stellate cells (HSCs)-T6, and to determine the possible underlying mechanisms. HSC-T6 cells were divided into three groups: Negative control group, oxidant-treated group and curcumin-treated group. Flow cytometry and spectrophotometry were used to measure the production of reactive oxygen species (ROS), and the levels of malondialdehyde (MDA) and glutathione (GSH). Immunocytochemistry and a radioimmunoassay were used to determine the expression of smooth muscle α-actin (α-SMA) and the secretion of extracellular matrix (ECM) molecules. In addition, western blotting and immunocytochemistry were used to determine the expression levels of nuclear factor-erythroid 2-related factor (Nrf2). Treatment with glucose oxidase (GO) significantly stimulated the formation of ROS and increased the production of MDA, as compared with the control cells; however, the production of GSH was only slightly increased. In addition, treatment with GO significantly promoted the expression of α-SMA and the secretion of ECM molecules. Conversely, treatment with curcumin significantly decreased the levels of ROS and MDA, and significantly increased the levels of GSH. Curcumin significantly inhibited the expression of α-SMA and decreased the secretion of ECM molecules. Furthermore, treatment with curcumin significantly increased the nuclear expression levels of Nrf2. These results indicated that curcumin may protect rat HSCs against oxidative stress and inhibit the GO-induced activation and secretion of ECM molecules in vitro. These effects were mediated by the upregulation of Nrf2 nuclear translocation.

CacyBP/SIP nuclear translocation induced by gastrin promotes gastric cancer cell proliferation.

AIM: To investigate the role of nuclear translocation of calcyclin binding protein, also called Siah-1 interacting protein (CacyBP/SIP), in gastric carcinogenesis. METHODS: The expression of CacyBP/SIP protein in gastric cancer cell lines was detected by Western blot. Immunofluorescence experiments were performed on gastric cancer cell lines that had been either unstimulated or stimulated with gastrin. To confirm the immunofluorescence findings, the relative abundance of CacyBP/SIP in nuclear and cytoplasmic compartments was assessed by Western blot. The effect of nuclear translocation of CacyBP/SIP on cell proliferation was examined using MTT assay. The colony formation assay was used to measure clonogenic cell survival. The effect of CacyBP/SIP nuclear translocation on cell cycle progression was investigated. Two CacyBP/SIP-specific siRNA vectors were designed and constructed to inhibit CacyBP/SIP expression in order to reduce the nuclear translocation of CacyBP/SIP, and the expression of CacyBP/SIP in stably transfected cells was determined by Western blot. The effect of inhibiting CacyBP/SIP nuclear translocation on cell proliferation was then assessed. RESULTS: CacyBP/SIP protein was present in most of gastric cancer cell lines. In unstimulated cells, CacyBP/SIP was distributed throughout the cytoplasm; while in stimulated cells, CacyBP/SIP was found mainly in the perinuclear region. CacyBP/SIP nuclear translocation generated a growth-stimulatory effect on cells. The number of colonies in the CacyBP/SIP nuclear translocation group was significantly higher than that in the control group. The percentage of stimulated cells in G1 phase was significantly lower than that of control cells (69.70% ± 0.46% and 65.80% ± 0.60%, control cells and gastrin-treated SGC7901 cells, P = 0.008; 72.99% ± 0.46% and 69.36% ± 0.51%, control cells and gastrin-treated MKN45 cells, P = 0.022). CacyBP/SIPsi1 effectively down-regulated the expression of CacyBP/SIP, and cells stably transfected by CacyBP/SIPsi1 were then chosen for further cellular assays. In CacyBP/SIPsi1 stably transfected cells, CacyBP/SIP was shown to be distributed throughout the cytoplasm, irregardless of whether they were stimulated or not. After CacyBP/SIP nuclear translocation was reduced, there had no major effect on cell proliferation, as shown by MTT assay. There had no enhanced anchorage-dependent growth upon stimulation, as indicated by colony formation in flat plates. No changes appeared in the percentage of cells in G0-G1 phase in either cell line (71.09% ± 0.16% and 70.86% ± 0.25%, control cells and gastrin-treated SGC7901-CacyBP/SIPsi1 cells, P = 0.101; 74.17% ± 1.04% and 73.07% ± 1.00%, control cells and gastrin-treated MKN45-CacyBP/SIPsi1 cells, P = 0.225). CONCLUSION: CacyBP/SIP nuclear translocation promotes the proliferation and cell cycle progression of gastric cancer cells.

Thioredoxin-like protein 2 is overexpressed in colon cancer and promotes cancer cell metastasis by interaction with ran.

AIMS: Our previous work identified thioredoxin-like protein 2 (Txl-2) as the target of the monoclonal antibody MC3 associated with colon cancer, but its underlying mechanisms remain poorly understood. Txl-2, a novel thioredoxin (Trx) and nucleoside diphosphate kinase family member, is alternatively spliced and gives rise to three different Txl-2 isoforms. In this study, Txl-2 expression in colon cancer, differential functions for Txl-2 isoforms in cell invasion and metastasis, and the downstream signaling were investigated. RESULTS: Txl-2 expression was elevated in colon cancer tissues compared to normal colonic tissues, with a high correlation between the histological grade and prognosis. Knockdown of Txl-2 expression significantly inhibited cancer cell motility, and the invasive and metastatic abilities of colon cancer cells. Interestingly, Txl-2 isoforms showed differential effects on cancer cell invasion and metastasis. Cell invasion and metastasis were significantly promoted by Txl-2b but inhibited by Txl-2c, while no obvious effect was observed for Txl-2a. Furthermore, a direct interaction was identified between Txl-2b and Ran, a Ras-related protein, by yeast two-hybrid assay and coimmunoprecipitation. PI3K pathway was found to be a major pathway mediating Txl-2b induced tumor invasion and metastasis. INNOVATION: The current study provides a novel biomarker and target molecule for the diagnosis and treatment of colon cancer and provides a novel paradigm to understand how alternative splicing functions in human cancer. CONCLUSION: Our findings demonstrate an elevated Txl-2 expression in colon cancer and that Txl-2b promotes cell invasion and metastasis through interaction with Ran and PI3K signaling pathway.

15-Hydroxyprostaglandin dehydrogenase is a tumor suppressor of human gastric cancer.

Cyclooxygenase-2 (COX-2), the key enzyme in prostaglandin synthesis, is often over-expressed in human gastric cancer. Recently, 15-hydroxyprostaglandin dehydrogenase [NAD+] (15-PGDH), the key enzyme in prostaglandin degradation, was found to be down-regulated in human gastric cancer tissues, but little is known about its role in gastric tumorigenesis. In this study, expression plasmids containing 15-PGDH siRNA were constructed and transfected into the gastric cancer cell line MKN45, which expresses endogenous 15-PGDH at a high level. The 15-PGDH gene was also transfected into the gastric cancer cell line SGC7901, which expresses endogenous 15-PGDH at a low level. When compared with the empty vector transfectant, MKN45 cells stably transfected with the 15-PGDH siRNA plasmid had a significantly increased proliferation rate. In contrast, SGC7901 cells stably transfected with the 15-PGDH cDNA had a significantly decreased growth rate. Furthermore, increased expression of 15-PGDH suppressed clone formation of gastric cancer cells in plate and soft agar colony formation assays in vitro and suppressed tumor formation in athymic nude mice in vivo. Stable silencing of 15-PGDH in gastric cancer cells also enhanced cell cycle entry in vitro. These results demonstrate for the first time that 15-PGDH acts as a tumor suppressor in human gastric cancer and provide further validation for 15-PGDH as a potential therapeutic target for human gastric cancer.

Identification of TRAK1 (Trafficking protein, kinesin-binding 1) as MGb2-Ag: a novel cancer biomarker.

The present study aimed to describe the characterization of an antibody MGb2 that reacts with an epitope on gastric cancer cells, and identification of MGb2 antigen (MGb2-Ag). Immunostaining revealed its distribution in human tissues and demonstrated that the positive rate of MGb2-Ag was 81.48% in gastric cancer, 100% in gastric signet-ring cell carcinoma and mucinous adenocarcinoma, 13.16% in precancerous conditions, and 0% in chronic superficial gastritis. Using Western blotting, immunoprecipitation and MALDI-TOF MS (matrix assisted laser desorption/ionization time-of-flight mass spectrometry), MGb2-Ag was identified as TRAK1 (Trafficking protein, kinesin-binding 1), a new molecular gained limited recognition. Both MGb2 and commercial anti-TRAK1 Ab recognized prokaryotic expressed TRAK1. Immunostaining characteristics of TRAK1 were identical with MGb2-Ag in continuous sections of paraffin-embedded tissues of gastric tissues. This is the first report that TRAK1/MGb2-Ag is a promising diagnostic marker for gastric cancer and may help to detect signet-ring cell carcinoma and mucinous adenocarcinoma.

Identification of the primary targets of carbamylation in bovine lens proteins by mass spectrometry.

PURPOSE: Carbamylation, an important post-translational modification of proteins, inevitably causes conformational changes of lens proteins. It may increase aggregation between crystallin molecules and disrupt the close packing required for transparency thus leading to cataract. The aim of this study was to isolate the primary targets of carbamylation in the lens and identify them by mass spectrometry. MATERIALS AND METHODS: Fresh intact bovine lenses were incubated with 100 mM potassium cyanate for 7 days. The proteins in the water-soluble fractions from the normal control and the cyanate-modified lens proteins were separated by two-dimensional (2-D) gel electrophoresis with identification after silver staining. Protein spots that differed between the normal and carbamylated groups were selected for further analysis using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). RESULTS: The 2-D gel results showed that the major lens proteins were in the section of pI 5-8, with relative molecular masses of 20-35 kDa, and changes in the carbamylated fraction like strings of beads indicating modification. The mass spectrometry analysis and a database search identified carbamylated proteins originating from alphaA-crystallin, betaB2- and gammaS-(betaS)-crystallins. CONCLUSIONS: These crystallins may be vulnerable proteins targeted by carbamylation. The accumulated aggregation and loss of chaperone activity may contribute to cataract formation.