Rho-ROCK Expression Predicts the Prognosis in Patients with T3/T4 Gastric Cancer

A small GTPase Rho protein and an effector ROCK have significant roles in cancer adhsion, metastasis, invasion, angiogenesis and cell mortality. We investigated the expressions of RhoA protein and ROCK-1 protein in 100 patients with macroscopically T3/T4 gastric cancer immunohistochemically. The expression of RhoA was detected in gastric cancer specimens from 39 patients and that of ROCK-1 in specimens from 30 patients. The clinicopathological characteristics of 21 tumors with co-expression of RhoA and ROCK-1 proteins (Rho/ROCK ON) were compared with those of the 79 remaining tumors (Rho/ROCK OFF). The percentage of lymph node metastasis positive cases in the Rho/ROCK ON group (81%) was higher than that in the Rho/ROCK OFF group (66%), but the difference was not significant (P = 0.183). However, the prognosis of the 21 patients with Rho/ROCK ON was significantly poorer than that of the 79 with Rho/ROCK OFF (P = 0.006). Our results indicate that the evaluation of the protein expression of RhoA and ROCK-1 is useful for predicting the prognosis in patients with T3/T4 gastric cancer

Suppression of osteoclastogenesis via α2-adrenergic receptors

The sympathetic nervous system is known to regulate osteoclast development. However, the involvement of α2-adrenergic receptors (α2-ARs) in osteoclastogenesis is not well understood. In the present study, their potential role in osteoclastogenesis was investigated. Guanabenz, clonidine and xylazine were used as agonists of α2-ARs, while yohimbine and idazoxan were employed as antagonists. Using RAW264.7 pre-osteoclast and primary bone marrow cells, the mRNA expression of the osteoclast-related genes nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), tartrate-resistant acid phosphatase (TRAP) and cathepsin K was evaluated following induction with receptor activator of nuclear factor κB ligand (RANKL). TRAP staining was also conducted to assess effects on osteoclastogenesis in mouse bone marrow cells in vitro. Administration of 5-20 µM guanabenz (P<0.01, for RANKL-only treatment), 20 µM clonidine (P<0.05, for RANKL-only treatment) and 20 µM xylazine (P<0.05, for RANKL-only treatment) attenuated RANKL-induced upregulation of NFATc1, TRAP and cathepsin K mRNA. Furthermore, the reductions in these mRNAs by 10 µM guanabenz and 20 µM clonidine in the presence of RANKL were attenuated by 20 µM yohimbine or idazoxan (P<0.05). The administration of 5-20 µM guanabenz (P<0.01, for RANKL-only treatment) and 10-20 µM clonidine (P<0.05, for RANKL-only treatment) also decreased the number of TRAP-positive multi-nucleated osteoclasts. Collectively, the present study demonstrates that α2-ARs may be involved in the regulation of osteoclastogenesis

The PI3K-Akt Pathway in SN-38-Induced Apoptosis in Human Gastric Cancer Cell Lines

SN-38, an active metabolite of a topoisomerase I inhibitor, CPT-11, exhibits a cytotoxic effect by inducing apoptosis in cancer cells. Phosphatidylinositol-3-OH kinase (PI3K)-Akt signaling is known to protect a variety of cells from apoptosis. The relationship between resistance to SN-38-induced apoptosis and the PI3K-Akt pathway in human gastric cancer cells is unknown. Here, we did an investigation using two gastric cancer cell lines, MKN1 and MKN45. Cell viability was determined by sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT) assay. Apoptosis was confirmed by fluorescence microscopy using Hoechst 33342 staining. Expression levels of phospho-Akt (pAkt) were determined by Western blotting. After being treated with SN-38, the populations of sub-G1 cells were induced by flow cytometry in 36.8% of MKN45 cells more frequently than in 13.5% of MKN1 cells. SN-38 inhibited the expression of pAkt dose-dependently in MKN45 cells, but not in MKN1 cells. In MKN1 cells, an additional pretreatment with the PI3K inhibitor, LY294002, led to the inhibition of pAkt expression and induced apoptosis. The results suggested that SN-38 induces apoptosis by decreasing PI3K-Akt survival signaling, the anti-apoptotic signals, in human gastric cancer cells. Akt inhibitor might be a useful anti-tumor agent in combination with CPT-11

A Predictive Factor of the Quality of Microarray Comparative Genomic Hybridization Analysis for Formalin-fixed Paraffin-embedded Archival Tissue

Utilizing formalin-fixed paraffin-embedded (FFPE) archival tissue, the most common form of tissue preservation in routine practice, for cytogenetic analysis using microarray comparative genomic hybridization (aCGH) remains challenging. We searched for a predictive factor of the performance of FFPE DNA in aCGH analysis. DNA was extracted from 63 FFPE archival tissue samples of various tissue types (31 breast cancers, 24 lung cancers, and 8 thyroid tumors), followed by aCGH analysis using high-density oligonucleotide microarrays. Tumor DNA from matched frozen samples and from FFPE samples after whole-genome amplification were also analyzed in 2 and 4 case, respectively. The derivative log ratio spread (DLRSpread) was used to assess the overall quality of each aCGH result. The DLRSpread correlated significantly with the double-stranded DNA ratio of tumor DNA, storage time, and the degree of labeling with Cy5 (P<0.0001; correlation coefficients=-0.796, 0.551, -0.481, respectively). Stepwise multiple linear regression analysis revealed that the double-stranded DNA ratio of tumor DNA is the most significant predictive factor of DLRSpread (regression coefficient=-0.4798; P=<0.0001). The cytogenetic profiles of FFPE and matched frozen samples showed good concordance. Although the double-stranded DNA ratios were increased after whole-genome amplification, the DLRSpread was not improved. The double-stranded DNA ratio can be used to predict the performance of aCGH analysis for DNA from FFPE samples. Using this quality metric, valuable FFPE archival tissue samples can be utilized for aCGH analysis