HTRA1 gene expression in gastric epithelial cells.

OBJECTIVES: To explore HtrA1 gene expression and its regulation in human gastric cancers. METHODS: The HtrA1 mRNA levels were examined by QPCR analysis and confirmed its expression with Northern blot analysis. The HtrA1 protein levels in all six gastric epithelial cell lines were investigated by Western blot analysis. Gene copy number was accessed and then sequenced the coding region from each mRNA in all six cell lines. The HtrA1 promoter region DNA methylation status was detected by using bisulfite sequencing analysis. Effect of decitabine and TSA on HTRA1 expression in gastric cancer cell line was determined by RTPCR. RESULTS: HIC analysis indicated that HtrA1 was highly expressed in normal epithelium, but dramatically down-regulated in gastric carcinoma tissues and variably expressed in tumor-adjacent tissues. HtrA1 gene expression was dramatically decreased in gastric carcinoma cells compared to non-tumorigenic counterparts. The HtrA1 gene loss in any of the 4 breast cancer cell lines was not detected. Total 14 CpGs in this region were all methylated in gastric cancer cells, whereas two normal cells, GES-1 and HFI-145, were having several unmethylated cytosines in this region. HtrA1 showed as ~Mr 44,000, Expression of HtrA1 protein was not observed in any of the four gastric cancer cell lines, BGC-823, MKN-45, SGC-7901and MKN-28. HtrA1 expression was observed in the HFI-145and GES-1 cell lines. CONCLUSIONS: The epigenetic silencing for HtrA1 gene expression could provide a possible strategy for re-activating HtrA1 gene expression in gastric cancer cells, thus facilitating further investigation of HtrA1's role in chemotherapy.

Prognostic significance of USP10 as a tumor-associated marker in gastric carcinoma.

Ubiquitin-specific protease 10 (USP10), a novel deubiquitinating enzyme, had been associated with growth of tumor cell. However, the role of USP10 in gastric cancer carcinogenesis had not been elucidated yet. The aim of this study was to investigate the expression level of USP10 in gastric carcinoma (GC) tissues and cell lines, then to evaluate the clinical significance of USP10 in GC patients. USP10, E-cadherin, Ki67 and p53 expressions were detected in 365 GC and 40 non-cancerous mucosa tissues by immunohistochemistry. Western blot for USP10 was performed on additional fresh GC tissues and GC cell lines. The expression level of USP10 in GC tissues was proved lower than that in non-cancerous mucosa tissues (p < 0.05). It was also lower in GC cell lines (AGS, BGC-823 and MKN45 cells) than that in gastric epithelial immortalized cell line (GES-1). Clinicopathological analysis showed that USP10 expression was negatively correlated with gastric wall invasion (p = 0.009), nodal metastasis (p = 0.002), and TNM stage (p = 0.000). In contrast, a positively correlation between the expression of USP10 and E-cadherin was found (p < 0.05), but there was no relationship proved between Ki67, p53 and USP10 (p > 0.05). On the Kaplan-Meier survival curves, we found poor prognosis in GC patients was associated with negative USP10 expression (p < 0.05). Moreover, USP10 expression was an independent prognostic factor for the overall survival in multivariate analysis. Our findings suggested that USP10 was an independent predictor of prognosis of GC patients.

Dendritic cells with CED-3 and CED-4 siRNA is effective in prolonging DC lifetime and suppresses tumor growth in gastric cancer.

BACKGROUND/AIMS: We investigated effects of CED-3 and CED-4-siRNA on prolonging dendritic cell life in vivo and in vitro. METHODOLOGY: The DCs were divided into three groups: pure-DC, siRNA and CED-3 and CED-4-siRNA. we performed anti-apoptosis assays for DCs with flow cytometry. The assay for cytotoxicity assay was performed in vitro by a standard chromium assay at various effector/target ratios. Percent-specific lysis was calculated. We injected three kinds of DCs from tail vein every 3 days, we calculated tumor volume control rate and tumor weight control rate with formula. RESULTS: The DC percentages of apoptosis of CED-3 and CED-4 siRNA group were (12.09±1.14)%. Tumor-specific CTL activity showed 82.1% specific lysis for CED-3 and CED-4-siRNA DC group and 39.4% and 40.2% specific lysis for pure DC and siRNA DC group respectively. The lysis of CED-3 and CED-4-siRNA group was higher than the any other groups (p<0.05).The experiment of transplantation tumor in BALB/C mice showed that CED-3 and CED-4-siRNA DC can inhibit mice with tumors in volume and weight. CONCLUSIONS: We found that vaccination with CED-3 and CED-4-siRNA was capable of prolonging the survival of antigen-expressing DCs, and generated a strong therapeutic effect in the treatment of gastric cancer.

RhoA and RhoC -siRNA inhibit the proliferation and invasiveness activity of human gastric carcinoma by Rho/PI3K/Akt pathway.

AIM: To evaluate the effects of adenovirus-mediated gene transfer of RhoA siRNA and RhoC siRNA on proliferation and invasion of SGC7901 cells by Rho/PI3K/Akt pathway. METHODS: Plasmid of RhoA siRNA and RhoC siRNA were constructed and transfected into SGC7901 cells. siRNA and LY294002 (PI3K inhibitor) were designed as the control group. The mRNA and protein expressions of RhoA and RhoC were respectively detected with RT-PCR and western blotting. In order to find out the changes of proliferation and invasion power of SGC7901 cell lines, we analyzed the data by MTT, Boyden chamber and evaluated apoptosis of cell with flow cytometry. We treated BALB/C nude mice with RhoA and RhoC-siRNA, and tumor control rate (%) in nude mice was calculated. RESULTS: RhoA and RhoC siRNA transfections specifically down-regulated the corresponding mRNA and protein levels in SGC7901 Cells. The experiment of permeated artificial basal membrane showed that the invasion power of SGC7901 cell lines are on the decline after treatment of Ad-RhoA and RhoC-siRNA (12.64 +/-3.27 vs 87.38 +/- 17.38, P < 0.05). The values of 490 nm wavelength light absorption were different in the five groups. The number of alive cells in the group of RhoA and RhoC-siRNA was lower than others in the 6(th) d (0.71 +/- 0.01 vs 3.82 +/- 0,11 P < 0.05). The apoptosis rate of transfected RhoA and RhoC-siRNA group with FACS were 19.07% +/- 1.78 and there were significant differences between treated and control groups (19.07 +/- 1.78% vs 1.23 +/- 0.11%, P < 0.01). The tumor transplantation experiment in BALB/C nude mice showed intratumoral injection of RhoA or RhoC siRNA can inhibit tumor growth. CONCLUSION: RhoA and RhoC siRNA gene therapy mediated by adenovirus may be useful for inhibiting growth and invasion of SGC7901 through a PI3K/Akt pathway. These results provide a novel therapeutic target in preventing gastric cancer cell invasion and metastasis.