CNTN-1 Upregulation Induced by Low-Dose Cisplatin Promotes Malignant Progression of Lung Adenocarcinoma Cells via Activation of Epithelial-Mesenchymal Transition.

Tumor metastasis and invasion are the main impediments to lung adenocarcinoma successful treatment. Previous studies demonstrate that chemotherapeutic agents can elevate the malignancy of cancer cells other than their therapeutic effects. In this study, the effects of transient low-dose cisplatin treatment on the malignant development of lung adenocarcinoma cells (A549) were detected, and the underlying epigenetic mechanisms were investigated. The findings showed that A549 cells exhibited epithelial-mesenchymal transition (EMT)-like phenotype along with malignant progression under the transient low-dose cisplatin treatment. Meanwhile, low-dose cisplatin was found to induce contactin-1 (CNTN-1) upregulation in A549 cells. Subsequently, we found that further overexpressing CNTN-1 in A549 cells obviously activated the EMT process in vitro and in vivo, and caused malignant development of A549 cells in vitro. Taken together, we conclude that low-dose cisplatin can activate the EMT process and resulting malignant progression through upregulating CNTN-1 in A549 cells. The findings provided new evidence that a low concentration of chemotherapeutic agents could facilitate the malignancy of carcinoma cells via activating the EMT process other than their therapeutic effects.

The phosphorylation and dephosphorylation switch of VCP/p97 regulates the architecture of centrosome and spindle.

The proper orientation of centrosome and spindle is essential for genome stability; however, the mechanism that governs these processes remains elusive. Here, we demonstrated that polo-like kinase 1 (Plk1), a key mitotic kinase, phosphorylates residue Thr76 in VCP/p97 (an AAA-ATPase), at the centrosome from prophase to anaphase. This phosphorylation process recruits VCP to the centrosome and in this way, it regulates centrosome orientation. VCP exhibits strong co-localization with Eg5 (a mitotic kinesin motor), at the mitotic spindle, and the dephosphorylation of Thr76 in VCP is required for the enrichment of both VCP and Eg5 at the spindle, thus ensuring proper spindle architecture and chromosome segregation. We also showed that the phosphatase, PTEN, is responsible for the dephosphorylation of Thr76 in VCP; when PTEN was knocked down, the normal spread of VCP from the centrosome to the spindle was abolished. Cryo-EM structures of VCPT76A and VCPT76E, which represent dephosphorylated and phosphorylated states of VCP, respectively, revealed that the Thr76 phosphorylation modulates VCP by altering the inter-domain and inter-subunit interactions, and ultimately the nucleotide-binding pocket conformation. Interestingly, the tumor growth in nude mice implanted with VCPT76A-reconstituted cancer cells was significantly slower when compared with those implanted with VCPWT-reconstituted cancer cells. Collectively, our findings demonstrate that the phosphorylation and dephosphorylation switch of VCP regulates the architecture of centrosome and spindle for faithful chromosome segregation.

Effects of root exudates on the activation and remediation of cadmium ion in contaminated soils.

To screen out plants with hyperaccumulation of heavy metals and explore the effects of root exudates on the phytoremediation in contaminated soils. The germination rates of five plants including Lolium perenne L. (L. perenne), Sorghum sudanense (Piper) Stapf. (S. sudanense), Pennisetum alopecuroides (L.) Spreng. (P. alopecuroides), Medicago sativa L. (M. sativa), and Trifolium repens L. (T. repens) in different concentrations of cadmium ion solution (0-100 mg/kg) were determined. The growth adaptability of these five plants under conditions of contaminated soils with the above cadmium ion concentrations was also evaluated. S. sudanense and P. alopecuroides had higher germination rates and better growth than the three other plants and were selected as the latter experimental varieties. The activation amounts of cadmium ion in soils were measured using AAS in the presence of three types of root secretions (citric acid, glycine, and maltose) with different concentrations (10-500 mmol/L). The activation amounts decrease in the following order: citric acid > glycine > maltose. The effect of these three root exudates on the removal of cadmium-contaminated soils in combination with S. sudanense and P. alopecuroides was also tested. For S. sudanense and P. alopecuroides, the maximum biomass and removal rate reaches the maximum at 100 mmol/L of citric acid. Conversely, low concentrations (approximately 10-50 mmol/L) of glycine and maltose are more effective for plant growth and phytoremediation. The addition of citric acid at 100 mmol/L and approximately 10-50 mmol/L of glycine and maltose can effectively promote the transfer of cadmium ion from roots to leaves and the accumulation of cadmium ion in leaves.

Curcumin Attenuates Asthmatic Airway Inflammation and Mucus Hypersecretion Involving a PPARγ-Dependent NF-κB Signaling Pathway In Vivo and In Vitro.

Asthma is characterized by airway inflammation and mucus hypersecretion. Curcumin possessed a potent anti-inflammatory property involved in the PPARγ-dependent NF-κB signaling pathway. Then, the aim of the current study was to explore the value of curcumin in asthmatic airway inflammation and mucus secretion and its underlying mechanism. In vivo, mice were sensitized and challenged by ovalbumin (OVA) to induce chronic asthma. Airway inflammation and mucus secretion were analyzed. In vitro, BEAS-2B cells were obtained. MCP-1, MUC5AC, and PPARγ expression and the phosphorylation of NF-κB p65 and NF-κB p65 DNA-binding activity were measured in both the lungs and BEAS-2B cells. shRNA-PPARγ was used to knock down PPARγ expression. We found that OVA-induced airway inflammation and mucus hypersecretion in mice, OVA and IL-4-induced upregulation of MCP-1 and MUC5AC, suppression of PPARγ, and activation and translocation of NF-κB p65 were notably improved by curcumin both in vivo and in vitro. Our data also showed that these effects of curcumin were significantly abrogated by shRNA-PPARγ. Taken together, our results indicate that curcumin attenuated OVA-induced airway inflammation and mucus hypersecretion in mice and suppressed OVA- and IL-4-induced upregulation of MCP-1 and MUC5AC both in vivo and in vitro, most likely through a PPARγ-dependent NF-κB signaling pathway.

Effects of Surface Charges on the Bactericide Activity of CdTe/ZnS Quantum Dots: A Cell Membrane Disruption Perspective.

The inhibitory effects of CdTe/ZnS quantum dots (QDs) modified with 3-mercaptopropionic acid (negatively charged) or cysteamine (positively charged) on the metabolic activity of Escherichia coli were investigated using biological microcalorimetry. Results show that the inhibitory ratio of positive QDs is higher than that of negative QDs. Transmission electron microscopy images indicate that QDs are prone to be adsorbed on the surface of E. coli. This condition disturbs the membrane structure and function of E. coli. Fluorescence anisotropy results demonstrate that positive QDs show a significant increase in the membrane fluidity of E. coli and dipalmitoylphosphatidylcholine (DPPC) model membrane. Furthermore, fluorescence anisotropy values of DPPC membrane in the gel phase decreased upon the addition of positive QDs. By contrast, anisotropy values in the liquid-crystalline phase are almost constant. The change in membrane fluidity is associated with the increased permeability of the membrane. Finally, the kinetics of dye leakage from liposomes demonstrate that the surface charge of QDs is crucial to the interaction between QDs and membrane.

Mycobacterium tuberculosis EIS gene inhibits macrophage autophagy through up-regulation of IL-10 by increasing the acetylation of histone H3.

Autophagy plays a crucial role in the progress of Mycobacterium tuberculosis (MTB) infection. Recently, MTB enhanced intracellular survival (EIS) protein was reported to be secreted from MTB cells and linked to the inhibition of autophagy and the intracellular persistence of the pathogen. Here, we investigated the mechanism of EIS-mediated inhibition of autophagy in a human phorbol myristate acetate (PMA)-treated THP-1 cell line as well as in murine macrophages. We confirmed that the presence of EIS led to the inhibition of rapamycin (Rapa)-induced autophagy, while IL-10 gene expression was increased and Akt/mTOR/p70S6K pathway was activated during the process. IL-10 gene silencing led to a significant recovery of EIS-mediated autophagy suppression and decreased activity of the Akt/mTOR/p70S6K pathway. IL-10 promoter activity was unaffected by EIS. Remarkably, EIS increased the acetylation level of histone H3 (Ac-H3), which binds to the SP1 and STAT3 region of the human IL-10 gene promoter sequence. Thus, EIS protein possibly increased IL-10 expression through the regulation of Ac-H3 of its promoter. Our data demonstrated that one possible mechanism of the MTB evasion of autophagy is that the EIS protein up-regulates IL-10 via Ac-H3 and thus activates Akt/mTOR/p70S6K pathway.

An intron SNP rs807185 in ATG4A decreases the risk of lung cancer in a southwest Chinese population.

Autophagy acts as a double-edged sword in cancer. Over the years, there has been growing evidence of the involvement of autophagy-related genes (ATGs) in the etiology and progression of cancer. Importantly, lung cancer is one of the most common cancers and represents the leading cause of cancer-related mortality in developing countries. The genomic variant has emerged as an important factor in the risk of lung cancer. Here, we hypothesize that the intron single-nucleotide polymorphism (SNP) of rs807185 in ATG4A is associated with the risk of lung cancer. In this case-control study, we genotyped the SNP rs807185 with PCR-restriction fragment length polymorphism. Our data suggest that the variant A allele frequency of rs807185 in controls is higher than that in cases (37.7 vs. 24.9%, P=0.006). The adjusted odds ratio is 1.989 (95% confidence interval 1.223-3.236). Compared with the wild T allele, the variant A allele of rs807185 in ATG4A is associated with a decreased risk of lung cancer (adjusted odds ratio=0.605, 95% confidence interval 0.456-0.803, P<0.001). Furthermore, stratified analysis in a recessive model suggests that the homozygous variant genotype (AA) of rs807185 could decrease the risk of lung cancer in smoking or nonsmoking groups. In conclusion, the variant of intron SNP rs807185 in ATG4A is associated significantly with a decreased risk of lung cancer in a southwest Chinese population. The results show that the variant rs807185 of ATG4A might be a protective factor for lung cancer.

The mechanism of acacetin-induced apoptosis on oral squamous cell carcinoma.

BACKGROUND: Acacetin (5,7-dihydroxy-40-methoxyflavone), present in safflower seeds, plants, flowers, Cirisium rhinoceros Nakai, has been reported to be able to exert anti-peroxidative, anti-inflammatory, anti-plasmodial, and anti-proliferative activities by inducing apoptosis and blocking the progression of cell cycles. OBJECTIVE AND DESIGN: The objective of this study is to investigate the mechanism of acacetin-induced apoptosis of oral squamous cell carcinoma cell line (HSC-3). RESULTS: Acacetin caused 50% growth inhibition (IC50) of HSC-3 cells at 25µg/mL over 24h in the MTT assay. Apoptosis was characterized by DNA fragmentation and increase of sub-G1 cells and involved activation of caspase-3 and PARP (poly-ADP-ribose polymerase). Maximum caspase-3 activity was observed with 100µg/mL of acacetin for 24h. Caspase-8 and -9 activation cascades mediated the activation of caspase-3. Acacetin caused reduction of Bcl-2 expression leading to an increase of the Bax:Bcl-2 ratio. It also caused a loss of mitochondrial membrane potential that induced release of cytochrome c into the cytoplasm. Pretreatment with casapse-3 (Z-DEVD-FMK), -8 (Z-IETD-FMK), and 9 inhibitor (z-LEHD-fmk) inhibited the acacetin-induced loss of mitochondrial membrane potential and release of cytochrome c. The mitogen-activated protein kinases (MAPKs) were activated by acacetin. Moreover, pretreating the cells with each of the caspase inhibitor or MAPKs specific inhibitors apparently inhibited acacetin-induced cytotoxicity of HSC-3 cells. CONCLUSION: In conclusion, acacetin induce the apoptosis of oral squamous cell carcinoma cell line, which is closely related to its ability to activate the MAPK-mediated signaling pathways with the subsequent induction of a mitochondria- and caspase-dependent mechanism. These results strongly suggest that acacetin might have cancer inhibition and therapeutic potential.

Significance of molecular markers in survival prediction of oral squamous cell carcinoma.

BACKGROUND: An accurate system for predicting the survival of patients with oral squamous cell carcinoma (OSCC) will be useful for deciding appropriate therapies. The prediction accuracy of prediction models can be improved by using molecular biomarkers. We constructed a nomogram for predicting the survival of patients with OSCC using clinical variables and molecular markers. METHODS: Protein 53 (p53), insulin-like growth factor II mRNA-binding protein 3 (IMP3), cyclooxygenase 2 (COX2), and HuR were localized immunohistochemistry in 96 patients with primary OSCC who underwent surgical resection between January 1994 and June 2003 at the Yonsei Dental Hospital in Seoul, Korea. RESULTS: On univariate and multivariate analysis, the expression of IMP3 was significantly associated with the risk of death. P53 was also significantly associated with survival of OSCC in the case of negative IMP3 and the prediction accuracy was improved by including these 2 factors in the prediction model. CONCLUSION: Survival in OSCC can be predicted more accurately by using biomarkers. The constructed nomogram predicted survival after treatment for an individual patient with OSCC, and it can be practically used as a tool to help decide which adjuvant treatment is most appropriate.

Chios mastic gum extracts as a potent antitumor agent that inhibits growth and induces apoptosis of oral cancer cells.

PURPOSE: The purpose was to investigate Chios mastic gum (CMG) extract as an potential anti-tumor agent for oral squamous cell carcinoma in vitro. METHODS: We designed a study to examine the effects of CMG extracts on growth of oral squamous cell carcinoma cell line, YD-10B and to determine whether the extracts could induce apoptosis through the activation of caspase-3, using the common chemotherapeutic agent Paclitaxel (Taxol, Bristol-Myers Squibb) as a control. RESULTS: MTT assay suggested that both CMG and Taxol inhibited the proliferation of YD-10B cells in a time and dose dependent manner. Moreover, 10 µg/mL of CMG and 50 µg/mL of Taxol caused fragmentation of the genomic DNA at 24 hour. Finally, 10 µg/mL of CMG and 50 µg/mL of Taxol caused cleavage of procaspase-3 in western blot analysis. CONCLUSIONS: These results suggest CMG's potential as an anti-tumor agent.

Rapid identification and detection of intracellular survival testing of mycobacterium smegmatis mc²155 that contains eis gene from mycobacterium tuberculosis by flow cytometry.

Mycobacterium tuberculosis is a facultative intracellular pathogen that has evolved the ability to survive and multiply within human macrophages. The enhanced intracellular survival (eis) gene (Rv2416c) from M. tuberculosis has been identified as a potential factor that can enhance the intracellular survival of Mycobacterium smegmatis in the macrophage cell line. However, the time requirements for intracellular survival testing of Mycobacterium using classical methodologies are still too long. In this study, we used M. smegmatis mc²155 that contains eis to develop and study a rapid method to test intracellular survival using flow cytometry. We demonstrated the success of this technique, which required only a few hours. This assay is rapid, accurate, and reproducible, and it would be valuable for the rapid detection of intracellular survival of mycobacteria.

Association between expression of embryonic lethal abnormal vision-like protein HuR and cyclooxygenase-2 in oral squamous cell carcinoma.

BACKGROUND: Increased cytoplasmic HuR expression has been noted in several cancer types, where it may contribute to the increased cyclooxygenase-2 (COX-2) expression observed during tumorigenesis and metastasis. METHODS: To assess the correlation between COX-2 and HuR in oral squamous cell carcinoma (OSCC), the expression patterns of HuR and COX-2 were assessed via immunohistochemistry analyses of 103 OSCC samples. RESULTS: Cytoplasmic HuR expression was significantly associated with COX-2 expression (p < .025) and lymph node metastasis (p < .050) and distant metastasis (p < .025). In multivariate analysis, cytoplasmic HuR expression was identified as an independent prognostic parameter for reduced overall survival. The inhibition of HuR expression by siRNA or leptomycin B (LMB) caused a reduction in the inducibility of COX-2 in oral cancer cells. CONCLUSION: Our results indicate that the cytoplasmic expression of HuR is associated with COX-2 expression in OSCCs and HuR can regulate COX-2 expression in oral cancer cell lines.

Insulin-like growth factor II mRNA-binding protein 3: a novel prognostic biomarker for oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is caused by multiple factors, including carcinogen exposure. Insulin-like growth factor II mRNA-binding protein 3 (IMP3) is highly expressed in various cancer cells but is rarely expressed in normal cells. We investigated whether IMP3 can be used as a prognostic biomarker for OSCC. METHODS: We performed immunohistochemistry and Western blotting to examine IMP3 expression in human tissues. We also investigated correlations among IMP3 expression, clinicopathologic factors, and overall survival. RESULTS: IMP3 was overexpressed in OSCC cells. The expression was correlated with high histologic grade, lymph node metastasis, and advanced tumor and clinical stages. Univariate analysis indicated that advanced clinical stages, lymph node metastases, and IMP3 expression were predictive factors for OSCC. Multivariate analysis showed that IMP3 expression was an independent prognostic indicator for OSCC. CONCLUSIONS: IMP3 expression was related to various clinicopathologic factors. IMP3 expression was an independent prognostic factor in patients with OSCC.

Construction of luciferase reporter gene vector for human MUC5AC gene promoter and analysis of its transcriptional activity.

OBJECTIVE: To clone the human mucin (MUC) 5AC gene promoter and construct its luciferase reporter vector for human MUC5AC gene and analyze its transcriptional activity. METHODS: The 1 348 bp DNA sequence at the human MUC5AC gene 5 end was analyzed by the Vector NTI software. After the target sequence from human A549 cells genomic DNA was amplified by PCR method, and the product of PCR was sequenced. By promoter deletion analysis, 3 promoter segments with different lengths were amplified by PCR, then the products were identified by DNA sequencing, and 4 promotor segments were inserted into pGL3- enhancer vectors. Site-specific mutagenesis technique was used to establish mutants of specificity protein (SP)-l and nuclear factor-kappa B (NF-kappaB) site in MUC5AC gene promoter. The relative luciferase activities were detected in the transfected A549 cells. RESULTS: Sequence analysis indicated that there were many cis-acting elements in the regions of 1 348 bp DNA sequence at the human MUC5AC gene 5 end. The 4 reporter gene vectors with promoter segments with different lengths were constructed successfully. Dual-luciferase assay revealed the 372 bp fragment including activity with the minimal fragment. Neutrophil elastase (NE) could increase the expression of luciferase reporter gene plasmid containing mutated NF-kappaB version (P<0.05 vs. control) of MUC5AC promoter in the transfected A549 cells. The induction by NE decreased markedly when the SP-l element in MUC5AC promoter were mutated. CONCLUSION: This research may provide an important basis for the further study of human MUC5AC gene promoter activity and regulation of gene expression. There is an up-regulative element of gene transcription in the region of -324 to -64 bp in MUC5AC gene upstream. SP-l site of the promotor mediates NE-induced MUC5AC expression in human A549 cells.

L1 cell adhesion molecule is a novel therapeutic target in intrahepatic cholangiocarcinoma.

PURPOSE: Intrahepatic cholangiocarcinoma (ICC), a highly malignant hepatobiliary cancer, has a poor prognosis and is refractory to conventional therapies. The aim of this study is to discover a novel molecular target for the treatment of ICC. EXPERIMENTAL DESIGN: To discover novel cancer-associated membrane antigens expressed in ICC cells, we generated monoclonal antibodies (mAb) by immunizing mice with intact ICC cell lines and screened for those that bind to the plasma membrane of ICC cells but not to normal cells. The mAb A10-A3 was selected and its target antigen was identified as the L1 cell adhesion molecule. Expression of L1 in ICC was evaluated by immunohistochemical analysis of tumor samples from 42 ICC patients. The functional significance of L1 expression in the tumor progression of ICC was investigated by L1 suppression, L1 overexpression, and antibody treatment. RESULTS: L1 was not expressed in normal hepatocytes and intrahepatic bile duct epithelium but highly expressed in 40.5% of ICC patients, remarkably at the invasive front of the tumors. Suppression of L1 with short hairpin RNA significantly decreased proliferation, migration, and invasion of ICC cells in vitro. Consistently, L1 overexpression in ICC cells enhanced proliferation, migration, invasion, and apoptosis resistance. In addition, L1 short hairpin RNA or anti-L1 mAb significantly reduced the tumor growth in nude mice bearing ICC xenograft. CONCLUSIONS: We identified that L1 is expressed in ICC. L1 plays an important role in the tumor progression of ICC by enhancing cell proliferation, migration, invasion, and survival. L1 may represent a novel therapeutic target for ICC.

L1 cell adhesion molecule is a novel independent poor prognostic factor of extrahepatic cholangiocarcinoma.

PURPOSE: Cholangiocarcinomas (CC) are associated with poor survival, but diagnostic markers and therapeutic targets have not yet been elucidated. We previously found aberrant expression of L1 cell adhesion molecule in intrahepatic CC and a role for L1 in the progression of intrahepatic CC. Here, we analyzed L1 expression in extrahepatic CC (ECC) and evaluated its prognostic significance. EXPERIMENTAL DESIGN: We examined L1 expression in tumors from 75 ECC patients by immunohistochemistry. We analyzed the correlations between L1 expression and clinicopathologic factors as well as patient survival. RESULTS: L1 was not expressed in normal extrahepatic bile duct epithelium but was aberrantly expressed in 42.7% of ECC tumors. High expression of L1 was detected at the invasive front of tumors and was significantly associated with perineural invasion (P < 0.01). Univariate analysis indicated that various prognostic factors such as histologic grade 3, advanced pathologic T stage and clinical stage, perineural invasion, nodal metastasis, and high expression of L1 were risk factors predicting patient survival. Multivariate analyses done by Cox's proportional hazards model showed that high expression of L1 (hazard ratio, 2.171; 95% confidence interval, 1.162-4.055; P = 0.015) and nodal metastasis (hazard ratio, 2.088; 95% confidence interval, 1.159-3.764; P = 0.014) were independent risk factors for patient death. CONCLUSIONS: L1 was highly expressed in 42.7% of ECC and its expression was significantly associated with perineural invasion. High expression of L1 and nodal metastasis were independent poor prognostic factors predicting overall survival in patients with ECC.

Identification of differentially expressed genes in papillary thyroid cancers.

PURPOSE: Techniques designed to identify differentially expressed genes (DEGs) in tumors have become important in modern pathology. Genefishing technique using the annealing control primer (ACP) system has recently been developed to screen for DEG transcripts. We tried to identify DEGs involved in papillary thyroid cancer (PTC) by using Genefishing technique. MATERIALS AND METHODS: We utilized a new differential display method, designated with Genefishing technique, to analyze DEGs in 21 cases of PTCs. RESULTS: Comparing the gene expression profiles between PTC and normal thyroid, we detected 17 genes that were differentially expressed in PTCs and performed cloning with sequencing in 10 genes. We confirmed the expression patterns of 2 DEGs by RT-PCR assay and identified the same results in 17 out of 21 (81%) PTCs. The 2 DEGs over-expressed in PTCs were identified as DC-STAMP and type I collagen A1. They are novel genes identified first in PTCs. CONCLUSION: We confirmed 2 DEGs in PTCs as DC-STAMP and type I collagen A1 by using Genefishing technique. Although the detailed functions of those 2 genes and their products remain to be determined, the genes will provide insights into mechanisms of carcinogenesis or tumor progression in PTCs.

L1 cell adhesion molecule as a predictor for recurrence in pulmonary carcinoids and large-cell neuroendocrine tumors.

Pulmonary neuroendocrine tumors are a distinct subset of neoplasms with indolent to aggressive behavior. This study was conducted to evaluate the prognostic role of L1 cell adhesion molecule (L1CAM) in pulmonary neuroendocrine tumors. We retrospectively analyzed L1 expression in 55 cases of completely resected carcinoids and large-cell neuroendocrine carcinomas, by the immunohistochemistry with monoclonal antibody A10-A3 against human L1. L1 immunoreactivity was detected in 34 (61.8%) of 55 specimens. There was a significant correlation between L1 expression and the World Health Organization classification of this tumor (Spearman rank correlation, rho=0.60, p<0.001). With median follow-up of 52.0 months, the 5-year survival rate for patients with low expression of L1 (<20% of tumor cells stained) was significantly better compared with those with high expression of L1 (82.6% vs. 43.7%, p=0.005). L1 was also a significant independent predictor of disease-free survival, and patients with high L1 expression have a higher risk for recurrence compared with those with low L1 expression (hazard ratio, 3.0; 95% confidence interval, 1.2-8.3; p=0.034). L1 expression is significantly associated with aggressiveness and further studies with larger samples are needed to validate potential prognostic value for pulmonary neuroendocrine tumors.

Chemokine receptor CXCR4 expression, function, and clinical implications in gastric cancer.

The chemokine receptor CXCR4 is associated with the biological behavior of cancer, but few studies have addressed the expression and function of CXCR4 in human gastric cancer and its impact on disease prognosis. We studied the expression of CXCR4 using RT-PCR, Western blotting, flow cytometry, and confocal microscopy in five gastric cancer cell lines. We also examined cell proliferation, migration, and anti-apoptotic activity in response to stromal cell-derived factor (SDF)-1alpha and evaluated SDF-1alpha/CXCR4 signaling pathways. Furthermore, we investigated the correlation between CXCR4 expression and the clinical features of 221 gastric cancer tissue samples. CXCR4 transcripts and proteins were detectable in all five gastric cancer cell lines. However, MKN-28, MKN-45, MKN-74, and SNU16 cells did not express membrane CXCR4. In contrast, KATO III cells expressed membrane CXCR4. In these cells, SDF-1alpha-induced migration was observed and was blocked by AMD3100, a specific inhibitor of CXCR4. SDF-1alpha induced rapid phosphorylation of Erk1/2 MAPK but did not promote phosphorylation of Stat3 or Akt. Gastric cancer tissue samples expressed CXCR4 with variable intensities. Strong CXCR4 expression was significantly associated with lymph node metastases (P=0.028) and higher stages III/IV (P=0.047), and further tended to be correlated with a reduced 5-year survival rate (42.6% vs. 53.9%; P=0.1). In conclusion, CXCR4 expression is associated with gastric cancer cell migration in vitro, and strong expression of CXCR4 by gastric cancer cells is significantly associated with lymphatic metastasis in patients with gastric cancer, suggesting that CXCR4 plays an important role during gastric cancer progression.

The ginsenoside metabolite compound K, a novel agonist of glucocorticoid receptor, induces tolerance to endotoxin-induced lethal shock.

Compound K (C-K), a protopanaxadiol ginsenoside metabolite, was previously shown to have immunomodulatory effects. Here, we describe a novel therapeutic role for C-K in the treatment of lethal sepsis through the modulation of Toll-like receptor (TLR) 4-associated signalling via glucocorticoid receptor (GR) binding. In mononuclear phagocytes, C-K significantly repressed the activation of TLR4/lipopolysaccharide (LPS)-induced NF-kappaB and mitogen-activated protein kinases (MAPKs), as well as the secretion of pro-inflammatory cytokines. However C-K did not affect the TLR3-mediated expression of interferon-beta or the nuclear translocation of IRF-3. C-K competed with the synthetic glucocorticoid dexamethasone for binding to GR and activated glucocorticoid responsive element (GRE)-containing reporter plasmids in a dose-dependent manner. In addition, the blockade of GR with either the GR antagonist RU486 or a siRNA against GR substantially reversed the anti-inflammatory effects of C-K. Furthermore, TLR4-dependent repression of inflammatory response genes by C-K was mediated through the disruption of p65/interferon regulatory factor complexes. Importantly, pre- or post-treatment with C-K significantly rescued mice from Gram-negative bacterial LPS-induced lethal shock by lowering their systemic inflammatory cytokine levels and by reversing the lethal sequelae of sepsis. Collectively, these results demonstrate that C-K, as a functional ligand of GR, regulates distinct TLR4-mediated inflammatory responses, and suggest a novel therapy for Gram-negative septic shock.