RASAL1 attenuates gastric carcinogenesis in nude mice by blocking RAS/ERK signaling.

Recent studies have suggested that the RAS protein activator like-1 (RASAL1) functions as a tumor suppressor in vitro and may play an important role in the development of gastric cancer. However, whether or not RASAL1 suppresses tumor growth in vivo remains to be determined. In the present study, we investigated the role of RASAL1 in gastric carcinogenesis using an in vivo xenograft model. A lentiviral RASAL1 expression vector was constructed and utilized to transfect the human poorly differentiated gastric adenocarcinoma cell line, BGC-823. RASAL1 expression levels were verified by quantitative real-time RT-PCR and Western blotting analysis. Then, we established the nude mice xenograft model using BGC-823 cells either over-expressing RASAL1 or normal. After three weeks, the results showed that the over-expression of RASAL1 led to a significant reduction in both tumor volume and weight compared with the other two control groups. Furthermore, in xenograft tissues the increased expression of RASAL1 in BGC-823 cells caused decreased expression of p-ERK1/2, a downstream moleculein the RAS/RAF/MEK/ERK signal pathway. These findings demonstrated that the over-expression of RASAL1 could inhibit the growth of gastric cancer by inactivation of the RAS/RAF/MEK/ERK pathway in vivo. This study indicates that RASAL1 may attenuate gastric carcinogenesis.

RASAL1 influences the proliferation and invasion of gastric cancer cells by regulating the RAS/ERK signaling pathway.

The aim of this study was to investigate the biological characteristics of the RASAL1 gene in a well-differentiated gastric cancer cell line MKN-28 and a poorly differentiated gastric cancer cell line BGC-823 cells, using RNA interference and gene transfection technology, respectively. MKN-28 cells were transfected with the shRNA of RASAL1 and BGC-823 cells were transfected with the pcDNA 3.1 plasmid vector containing RASAL1. RT-PCR and western blotting were then used to detect the expression of RASAL1 mRNA and protein. The activities of RAS and extracellular signal-regulated kinase 1/2 were analyzed by the pull-down method and western blotting. The proliferate capacity, apoptosis rate, invasive and migratory potentials of MKN-28 or BGC-823 cells were also measured by Cell Counting Kit-8 cell proliferation assay, propidium iodide/Annexin V staining coupled with flow cytometry, and transwell chamber assays, respectively. Measurement of RASAL1 mRNA and protein expression in two cells revealed successful transfection of the shRNA of RASAL1 and RASAL1-pcDNA3.1 plasmid into these two cells. Moreover, decreased expression of RASAL1 in MKN-28 cells resulted in increased expression of RAS-GTP and p-ERK1/2. Interestingly, decreased expression of RASAL1 inhibited apoptosis and facilitated cell proliferation, invasion and migration. The increased expression of RASAL1 in BGC-823 cells caused declined expression of RAS-GTP and p-ERK1/2, as well as promoted apoptosis and restrained cell proliferation, invasion and migration. The down-regulation of RASAL1 promoted the proliferation, invasion and migration of gastric cancer MKN-28 cells, and up-regulation of RASAL1 inhibited the proliferation, invasion and migration of BGC-823 gastric cancer cells by regulating the RAS/ERK signaling pathway. Thus, our results suggest that RASAL1 may play an important role as a tumor suppressor gene in gastric cancer.

Hypermethylation and clinicopathological significance of RASAL1 gene in gastric cancer.

BACKGROUND: Recent studies have suggested that expression of the RAS protein activator like-1 gene (RASAL1) is decreased in gastric carcinoma tissues and cell lines, indicated a role in tumorigenesis and development of gastric cancer. Reduced expression of RASAL1 could result in aberrant increase of activity of RAS signaling pathways in cancer cells. However, the exact mechanism which induces down-regulation of the RASAL1 gene remains unclear. This study aimed to determine the methylation status and regulation of RASAL1 in gastric cancer. MATERIALS AND METHODS: Using the methylation-specific polymerase chain reaction (MSP), the methylation status of CpG islands in the RASAL1 promoter in gastric cancers and paired adjacent non-cancerous tissues from 40 patients was assessed and its clinicopathological significance was analyzed. The methylation status of RASAL1 in gastric cancer lines MKN-28, SGC-790l, BGC-823, as well as in normal gastric epithelial cell line GES-l was also determined after treatment with a DNA methyltransferase inhibitor, 5-aza-2'-doexycytidine (5-Aza-CdR). RAS activity (GAS-GTP) was assessed through a pull-down method, while protein levels of ERK1/2, a downstream molecule of RAS signaling pathways, were determined by Western blotting. RESULTS: The frequencies of RASAL1 promoter methylation in gastric cancer and paired adjacent non-cancerous tissues were 70% (28/40) and 30% (12/40) respectively (P<0.05). There were significantly correlations between RASAL1 promoter methylation with tumor differentiation, tumor size, invasive depth and lymph node metastasis in patients with gastric cancer (all P<0.05), but no correlation was found for age or gender. Promoter hypermethylation of the RASAL1 gene was detected in MKN-28, SGC-790l and BGC-823 cancer cells, but not in the normal gastric epithelial cell line GES-1. Elevated expression of the RASAL1 protein, a decreased RAS-GTP and p-ERK1/2 protein were detected in three gastric cancer cell lines after treatment with 5-Aza-CdR. CONCLUSIONS: Aberrant hypermethylation of the RASAL1 gene promoter frequently occurs in gastric cancer tissues and cells. In addition, the demethylating agent 5-Aza-CdR can reverse the hypermethylation of RASAL1 gene and up-regulate the expression of RASAL1 significantly in gastric cancer cells in vivo. Our study suggests that RASAL1 promoter methylation may have a certain relationship with the reduced RASAL1 expression in gastric cancer.