A decision tree-based combination of ezrin-interacting proteins to estimate the prognostic risk of patients with esophageal squamous cell carcinoma.

Our previous studies have highlighted the importance of ezrin in esophageal squamous cell carcinoma (ESCC). Here our objective was to explore the clinical significance of ezrin-interacting proteins, which would provide a theoretical basis for understanding the function of ezrin and potential therapeutic targets for ESCC. We used affinity purification and mass spectrometry to identify PDIA3, CNPY2, and STMN1 as potential ezrin-interacting proteins. Confocal microscopy and coimmunoprecipitation analysis further confirmed the colocalization and interaction of ezrin with PDIA3, CNPY2, and STMN1. Tissue microarray data of ESCC samples (n=263) showed that the 5-year overall survival (OS) and disease-free survival (DFS) were significantly lower for the CNPY2 (OS, P=.003; DFS, P=.011) and STMN1 (OS, P=.010; DFS, P=.002) high-expression groups compared with the low-expression groups. By contrast, overexpression of PDIA3 was significantly correlated with favorable survival (OS, P<.001; DFS, P=.001). Cox regression demonstrated the prognostic value of PDIA3, CNPY2, and STMN1 in ESCC. Furthermore, decision tree analysis revealed that the resulting classifier of both ezrin and its interacting proteins could be used to better predict OS and DFS of patients with ESCC. In conclusion, a signature of ezrin-interacting proteins accurately predicts ESCC patient survival or tumor recurrence.

Clinical and experimental studies regarding the expression and diagnostic value of carcinoembryonic antigen-related cell adhesion molecule 1 in non-small-cell lung cancer.

BACKGROUND: Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a multifunctional Ig-like cell adhesion molecule that has a wide range of biological functions. According to previous reports, serum CEACAM1 is dysregulated in different malignant tumours and associated with tumour progression. However, the serum CEACAM1 expression in non-small-cell lung carcinomas (NSCLC) is unclear. The different expression ratio of CEACAM1-S and CEACAM1-L isoform has seldom been investigated in NSCLC. This research is intended to study the serum CEACAM1 and the ratio of CEACAM1-S/L isoforms in NSCLC. METHODS: The expression of the serum CEACAM1 was determined by enzyme-linked immunosorbent assay. The protein expression and the location of CEACAM1 in tumours were observed by immunohistochemical staining. The CEACAM1 mRNA levels in tumour and normal adjacent tissues were measured using quantitative real-time PCR, and the expression patterns and the rate of CEACAM1-S and CEACAM1-L were analysed by reverse transcription-PCR. RESULTS: Serum CEACAM1 levels were significantly higher in NSCLC patients compared with that from normal healthy controls (P <0.0001). 17 patients (81%) among 21 showed high expression of CEACAM1 by immunohistochemical staining. Although no significant differences were found between tumour and normal tissues on mRNA expression levels of CEACAM1 (P >0.05), the CEACAM1-S and the CEACAM1-S/L (S: L) ratios were significantly higher in tumour than normal tissues (P <0.05). CONCLUSIONS: Our data indicated that the serum levels of CEACAM1 could discriminate lung cancer patients from health donors and that CEACAM1 might be a useful marker in early diagnosis of NSCLC. Moreover, our results showed that the expression patterns of CEACAM1 isoforms could be changed during oncogenesis, even when total CEACAM1 in tumour tissues did not show significant changes. Our study suggested that the expression ratios of CEACAM1-S/CEACAM1-L might be a better diagnostic indicator in NSCLC than the quantitative changes of CEACAM1.

DACT2 is a candidate tumor suppressor and prognostic marker in esophageal squamous cell carcinoma.

In animals ranging from fish to mice, the function of DACT2 as a negative regulator of the TGF-ß/Nodal signal pathway is conserved in evolution, indicating that it might play an important role in human cancer. In this study, we showed that tumors with higher DACT2 protein level were correlated with better differentiation and better survival rate in patients with esophageal squamous cell carcinoma. Restored expression of DACT2 significantly inhibited growth, migration, and invasion of ESCC cells in vitro, and reduced tumorigenicity in vivo. Furthermore, when DACT2 expression was restored, the activity of TGF-ß/SMAD2/3 was suppressed via both proteasome and lysosomal degradation pathways, leading to F-actin rearrangement that might depend on the involvement of cofilin and ezrin-redixin-moesin (ERM) proteins. Taken together, we propose here that DACT2 serves as a prognostic marker that reduces tumor cell malignancy by suppressing TGF-ß signaling and promotes actin rearrangement in ESCC.

The mechanism underlying the effects of the cell surface ATP synthase on the regulation of intracellular acidification during acidosis.

The F1F0 ATP synthase has recently become the focus of anti-cancer research. It was once thought that ATP synthases were located strictly on the inner mitochondrial membrane; however, in 1994, it was found that some ATP synthases localized to the cell surface. The cell surface ATP synthases are involved in angiogenesis, lipoprotein metabolism, innate immunity, hypertension, the regulation of food intake, and other processes. Inhibitors of this synthase have been reported to be cytotoxic and to induce intracellular acidification. However, the mechanisms by which these effects are mediated and the molecular pathways that are involved remain unclear. In this study, we aimed to determine whether the inhibition of cell proliferation and the induction of cell apoptosis that are induced by inhibitors of the cell surface ATP synthase are associated with intracellular acidification and to investigate the mechanism that underlines the effects of this inhibition, particularly in an acidic tumor environment. We demonstrated that intracellular acidification contributes to the cell proliferation inhibition that is mediated by cell surface ATP synthase inhibitors, but not to the induction of apoptosis. Intracellular acidification is only one of the mechanisms of ecto-ATP synthase-targeted antitumor drugs. We propose that intracellular acidification in combination with the inhibition of cell surface ATP generation induce cell apoptosis after cell surface ATP synthase blocked by its inhibitors. A better understanding of the mechanisms activated by ecto-ATP synthase-targeted cancer therapies may facilitate the development of potent anti-tumor therapies, which target this enzyme and do not exhibit clinical limitations.

Antitumor activity of histone deacetylase inhibitor trichostatin A in osteosarcoma cells.

BACKGROUND: Histone deacetylase (HDAC) inhibitors have been reported to induce cell growth arrest, apoptosis and differentiation of tumor cells. The present study aimed to examine the effects of trichostatin A (TSA), one such inhibitor, on the cell cycle, apoptosis and invasiveness of osteosarcoma cells. METHODS: MG- 63 cells were treated with TSA at various concentrations. Then, cell growth and apoptosis were determined by 3-(4, 5-dimethyl-2-thiazolyl)-2H-tetrazolium bromide (MTT) and TUNEL assays, respectively; cell cycling was assessed by flow cytometry; invasion assays were performed with the transwell Boyden Chamber system. RESULTS: MTT assays revealed that TSA significantly inhibited the growth of MG-63 cells in a concentration and time dependent manner. TSA treated cells demonstrated morphological changes indicative of apoptosis and TUNEL assays revealed increased apoptosis of MG-63 cells after TSA treatment. Flow cytometry showed that TSA arrested the cell cycle in G1/G2 phase and annexin V positive apoptotic cells increased markedly. In addition, the invasiveness of MG-63 cells was inhibited by TSA in a concentration dependent manner. CONCLUSION: Our findings demonstrate that TSA inhibits the proliferation, induces apoptosis and inhibits invasiveness of osteosarcoma cells in vitro. HDAC inhibitors may thus have promise to become new therapeutic agents against osteosarcoma.

A monoclonal antibody (Mc178-Ab) targeted to the ecto-ATP synthase ß-subunit-induced cell apoptosis via a mechanism involving the MAPKase and Akt pathways.

Ecto-ATP synthase has been considered to be an effective target for cancer recently. As inhibitors of ecto-ATP synthase were found to be cytotoxic for tumor cells, a monoclonal antibody (Mc178-Ab) against ecto-ATP synthase was generated in our previous study that exhibited both anti-angiogenic and anti-tumorigenic effects. However, the mechanism of action of Mc178-Ab and its downstream pathways for anti-tumor effects remain unclear. In this research, we intended to investigate the mechanism of the anti-tumor action of Mc178-Ab. The expressions of cell surface ATP synthase on A549 and CHO cells were confirmed by flow cytometry and confocal microscope. Proliferation and apoptosis were examined after the treatment with Mc178-Ab. In order to examine the activity of ecto-ATP synthase changed by Mc178-Ab, extracellular ATP generation and intracellular pH levels were assessed. The phosphorylation of the signaling molecules, MAPKase and Akt, was analyzed by western blot. Cell proliferation was blocked, and apoptosis was induced in A549 cells treated with Mc178-Ab, as determined by MTT assay and flow cytometry analysis of Annexin-V/PI staining separately. The intracellular pH level and extracellular ATP generation were also decreased after Mc178-Ab treatment. Finally, western blot data revealed that the phosphorylation of JNK and p38 was increased, while the phosphorylation of ERK and Akt was decreased in A549 cells treated with Mc178-Ab. Compared with A549 cells, Mc178-Ab had less effect on CHO cells. The decreased intracellular pH levels and the altered concentration of extracellular ATP may contribute to the mechanisms of the effect of Mc178-Ab on A549 and CHO cells. The results also suggested that the anti-tumor effect of Mc178-Ab was associated with MAPKase and Akt pathways.

CD44 mediates oligosaccharides of hyaluronan-induced proliferation, tube formation and signal transduction in endothelial cells.

Oligosaccharides of hyaluronan (o-HA) can induce angiogenesis and the growth and tube formation of vascular endothelial cells (ECs) in particular. As the major o-HA receptor, CD44 has been implicated in EC function, but its role in mediating o-HA-induced EC proliferation and tube formation remains unclear. In this study, we investigated the role of CD44 in o-HA-induced proliferation and tube formation of human umbilical vein endothelial cells (HUVECs) and explored the molecular mechanisms underlying the angiogenesis process. A CD44 siRNA was delivered into HUVECs by electroporation and o-HA-induced proliferation and tube formation capacity of CD44-silenced or control HUVECs were assessed by methylthiazolyldiphenyl-tetrazolium bromide (MTT) and Matrigel assays. Furthermore, the changes in Src, focal adhesion kinase (FAK) and extracellular signal-regulated kinase1 and 2 (ERK1/2) phosphorylation, as well as the expression of c-jun and c-fos were examined by Western blot and realtime-polymerase chain reaction assays. Our results demonstrated that 10 µg/mL o-HA obviously induced the proliferation and tube formation in HUVECs, and stimulated the phosphorylation of Src, FAK and ERK1/2 and upregulation of c-jun and c-fos, which could be inhibited by CD44 silencing. Altogether our data suggest that CD44 functions to initiate tyrosine phosphorylation of Src, FAK and ERK1/2, and upregulates the expression of c-jun and c-fos, thus mediating o-HA-induced proliferation and tube formation in HUVECs.

[Src kinase-MAPK signal pathway plays a role in proliferation of endothelial cells induced by o-HA].

To investigate the effect of hyaluronan oligosaccharides (o-HA) on endothelial cell (EC) proliferation and the possible mechanism involved. The cell proliferation was determined by cell counting and flow cytometer, and the phosphorylation of Src kinase and ERK-1/2 as well as the expression of cyclin D1 were assayed by western blot. o-HA at concentration of 10 microg/ml caused a significantly increase in both cell cycle and cell number of EC. With increasing time and amount of o-HA of exposure, there was no further increase in the growth of cells. The cell proliferation started to be significant at 12 hr and reached peak at 72 hr. At the same time,the phosphorylation of Src kinase and ERK-1/2 was enhanced after treated with l microg/ml of o-HA at 5 min and the expression of cyclin D1 was enhanced by treating PIEC cells with o-HA at 3 hr. o-HA may increase EC growth by stimulating the Src kinase and MAPK signal pathway and thus promote the proliferation of PIEC cells,in which the regulation of cyclin D1 expression may be involved.