27-Hydroxycholesterol enhanced osteoclastogenesis in lung adenocarcinoma microenvironment.

27-Hydroxycholesterol (27-HC) has been implicated in the pathological process of estrogen receptor positive breast cancer. However, the role of 27-HC in lung adenocarcinoma is still unclear. Because bone metastasis is a main reason for the high mortality of lung adenocarcinoma, this study aimed to investigate the effect of 27-HC on osteoclastogenesis in lung adenocarcinoma microenvironment. The results showed that the conditioned media (CM) from lung adenocarcinoma cells cocultured with macrophages promoted osteoclast differentiation, which was enhanced by 27-HC. Further investigation showed that CM inhibited miR-139 expression and promoted c-Fos expression. Luciferase reporter assay identified c-Fos as a direct target of miR-139. CM also induced the expression and nuclear translocation of NFATc1 and STAT3 phosphorylation, which was enlarged by 27-HC but was attenuated by miR-139. Coimmunoprecipitation assay demonstrated that 27-HC increased the interaction between NFATc1 and phosphorylated STAT3, which was restricted by miR-139. Chromatin immunoprecipitation assay showed that pSTAT3 could bind to the promoter of c-Fos, c-Fos could bind to the promoter of NFATc1, and both pSTAT3 and NFATc1 could bind to the promoter of Oscar, which were enlarged by 27-HC but were blocked by miR-139. Knockdown of c-Fos mimicked the effect of miR-139. These results suggested that CM, especially containing 27-HC, promoted osteoclastogenesis by inhibiting miR-139 expression and activating the STAT3/c-Fos/NFATc1 pathway.

KIAA0101 inhibition suppresses cell proliferation and cell cycle progression by promoting the interaction between p53 and Sp1 in breast cancer.

KIAA0101 functions as a regulator of centrosome number in breast cancer. Here, we identify the role of KIAA0101 in breast cancer cell proliferation and cell cycle progression. KIAA0101 knockdown significantly inhibited cell growth, colony formation and G1/S phase transition. Further investigation indicated that KIAA0101 silencing suppressed the expression of CCNE2, CDK6 and CDKN1A. Luciferase reporter assay and ChIP assay demonstrated that Sp1 positively regulated the transcription of CCNE2, CDK6 and CDKN1A. KIAA0101 knockdown promoted the interaction between p53 and Sp1, inhibiting the transcriptional activation of Sp1 on CCNE2, CDK6 and CDKN1A. Knockdown of p53 counteracted the inhibitory effect of KIAA0101 knockdown on breast cancer cells proliferation and cell cycle progression while Sp1 knockdown mimicked the effect of KIAA0101 knockdown. These results suggested that KIAA0101 knockdown suppressed the cell proliferation and cell cycle progression by promoting the formation of p53/Sp1 complex in breast cancer.

A pharmacokinetic study of Isatin in Beagles' bodies.

Isatin are marine active drugs that exert anti-cancer effects, have a cancer-prevention function, and possess many pharmacological activities. The study aimed to examine the pharmacokinetics of a single intravenous injection and oral medication of Isatin given to Beagles. Nine male and nine female Beagles were injected with 30 mg/kg of 2,3-indole quinones. The animals were divided into 3 groups (n=6 per group) and lavaged with a dose of 15, 30 and 60 mg/kg, respectively. Blood samples were collected prior to the medicine delivery (0 h) and 0.083, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h post-medicine delivery. The blood plasma samples were analyzed using the liquid chromatography-mass spectrometry (MS)/MS method following pretreatment for the protein precipitation. Pharmacokinetics software was applied to calculate relevant pharmacokinetic parameters through the atrioventricular model. The drug concentration in plasma decreased rapidly following the intravenous injection of Isatin. After 8 h, the prototype drugs could not be tested in the plasma and only trace amounts of drugs were tested in one dog, which was considered to be an endogenous drug. Indole quinone was absorbed following lavage into Beagles and peaked in <1 h, and the drug concentration in the plasma decreased rapidly. After 8 h, the prototype drugs could not be tested in the plasma. The elimination of the two drugs in the body had no evident gender differences. In conclusion, Isatin is rapidly absorbed in bodies of Beagles. Within the dose range of 15-60 mg/kg, no linear relationship was observed for the increase in Cmax and AUC0-t values with the increased dose.

Lentivirus-mediated gene silencing of NOB1 suppresses non-small cell lung cancer cell proliferation.

NIN/RPN12 binding protein 1 (NOB1p) encoded by NOB1 has been found to be an essential factor in 26S proteasome biogenesis which participates in protein degradation. However, the functions of NOB1 in non-small cell lung cancer cells are largely unknown. In the present study, lentivirus-mediated NOB1 shRNA transfection in two non-small cell lung cancer cell lines (A549 and H1299) was accomplished, as determined by fluorescence imaging. Downregulation of NOB1 expression was confirmed by real-time PCR and western blotting. NOB1 silencing resulted in a significant decline in the proliferation and colony formation capability of non-small cell lung cancer cells. Moreover, flow cytometry showed that A549 cells were arrested in the G0/G1 phase of the cell cycle after NOB1 suppression. Furthermore, depletion of NOB1 resulted in a significant decrease in CDK4 and cyclin D1 expression. These results suggest that NOB1 may act as an important regulator in non-small cell lung cancer growth and could be a therapeutic target of non­small cell lung cancer.

The prognostic value of ratio-based lymph node staging in resected non-small-cell lung cancer.

INTRODUCTION: Assessment of lymph node status is a critical issue in the surgical management of non-small-cell lung cancer (NSCLC). We sought to determine the prognostic value of metastatic lymph node ratio (LNR) in patients with radical surgery for NSCLC. METHODS: We abstracted data from 480 consecutive patients undergoing radical surgery for NSCLC between 2006 and 2008 in our institution. Kaplan-Meier estimated the survival function using the number of metastatic lymph node (MLN) and LNR as categorized variables. The prognostic value of age, sex, smoking status, location of tumor, histology, pathology T stage, pathology N stage, surgical procedure, chemotherapy, MLN, and LNR were assessed using a multivariate Cox proportional hazards model for overall survival (OS) and disease-free survival (DFS). RESULTS: The median numbers of examined lymph nodes and MLNs were 15 and 5, respectively. Optimal cutpoints of the LNR were calculated as 0, 0 to 0.35, and greater than 0.35. Patients with higher LNR were associated with worse OS and DFS in the whole series, whereas there was no significant difference in the OS and DFS of those patients classified as pathology N2. A multivariate analysis showed that the LNR staging, smoking status, and chemotherapy were revealed to be independent prognostic factors. CONCLUSIONS: LNR is an independent predictor of survival in patients with NSCLC undergoing radical resection; the prognostic significance is more valuable in patients classified as pathology N1.

MicroRNA-34b functions as a tumor suppressor and acts as a nodal point in the feedback loop with Met.

MicroRNAs (miRNAs), as a class of naturally occurring small non-coding RNAs, play profound and pervasive roles in cancer initiation and progression. Extensive decrease in miRNA levels are frequently observed in human cancers, indicating that miRNAs may function intrinsically in tumor suppression. However, the underlying mechanisms of miRNA interactions with cellular pathways are still unclear. The expression of miR-34b in non-small cell lung cancer (NSCLC) tissues was detected using quantitative real-time PCR. The relations between miR-34b expression levels and pathological stage or lymph node metastasis were assessed using the Spearman correlation test. For in vitro studies, lung cancer cells were transfected with double stranded synthetic miRNA mimics (syn-hsa-miR-34b miScript miRNA) and scrambled controls. Immunohistochemistry was used to validate the related downstream proteins of miR-34b. The expression of miR-34b was lower in NSCLC tissues compared to that in pericarcinous tissues of lung cancer. Additionally, the Spearman correlation test showed that lower miR-34b expression was correlated with higher lymph node metastasis. In vitro gain-of-function experiments indicated that miR-34b suppressed cell proliferation by inducing cell apoptosis. IHC results showed association between lower miR-34b and overexpression of phospho-Met, p53 (phospho S392) and Mdm2. Consistent with the opposing correlation between the expression of miR-34b and lymph node metastasis in NSCLC, miR-34b may play an important role in NSCLC progression. Furthermore, miR-34b downregulates Met, with subsequent changes of downstream p53 (phospho S392) and Mdm2, and inversely p53 upregulates miR-34b in a feedback loop, which provides new insights into the roles of miR-34 family members in the regulation of signaling pathways of NSCLC.

Expression of ß-arrestin 1 in gastric cardiac adenocarcinoma and its relation with progression.

OBJECTIVE: Arrestins act as mediators of G protein-coupled receptor (GPCR) desensitization and trafficking, also actin as a scaffold for many intracellular signaling network. The role that ß-arrestin 1 plays in gastric cardiac adenocarcinoma (GCA) and its clinicopathologic significance are untouched. METHODS: Fifty patients with gastric cardiac adenocarcinoma were retrospectively enrolled and ß-arrestin 1 was detected using immunohistochemistry in tissue samples. RESULTS: Nuclear expression of ß-arrestin 1 was observed in 78% of GCA samples (39/50) and cytoplasmic expression in 70% (35/50). ß-arrestin 1 could be found in both nucleus and cytoplasm of 54% GCA (27/50) or in either of them in 94% (47/50). ß-arrestin 1 protein positivity in well/ moderately differentiated carcinomas was significantly higher than that in poorly differentiated carcinomas (P=0.005). We found increased expression of ß-arrestin 1 in cytoplasm was correlated with lymph nodal metastasis (P=0.002) and pathological lymph nodal staging (P=0.030). We also found ß-arrestin 1 to be over-expressed in glandular epithelia cells of mucinous adenocarcinoma, a tumour type associated with an adverse outcome of gastric cardiac adenocarcinoma (P=0.022). CONCLUSION: ß-arrestin 1 is over-expressed in the nucleus and/or cytoplasm of gastric cardiac adenocarcinoma. However, ß-arrestin 1 has no relationship with the prognosis of gastric cardiac adenocarcinoma (P>0.05). Our data imply that ß-arrestin 1 in cytoplasm may be involved in differentiation and metastasis of gastric cardiac adenocarcinoma.