Effects Of Caspase-8 Promoter Methylation Status On TRAIL Antitumor Activity Of Human Gastric Cancer Cells: A Study Of MSP, RT-PCR And MTT

Objective Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a large variety of cancer cells but not in most normal human cells. This feature makes TRAIL, a potential antitumor agent. Methylation of the promoter regions of CpG-rich sites in Caspase-8, an apoptosis inducing cysteine protease is the major mechanism for the silencing of this gene in tumors, and may contribute to an increased mutation and allelic loss of this gene. Recent reports indicated that resistance to TRAIL-induced apoptosis in tumor cells correlates with a loss of caspase-8 expression. Deregulation of caspase-8 expression is through DNA methylation as well as through gene deletion. Several preclinical and clinical trials have been developed to use DNA methylation inhibitors, such as 5-aza-2'-deoxycytidine (5-Aza-CdR) in attempts to reactivate silenced genes in human cancers. 5-Aza-CdR can binding to the actual DNA (cytosine-5) methyltransferases (DNMTs) to reverse hypermethylation of caspase-8 resulting in restoration of caspase-8 expression and to restore sensitivity for TRAIL in various tumors in vivo. However, whether methylation of caspase-8 influences the antitumor activity of TRAIL in gastric cancer cell has not been extensively studied. This study was designed to explore effects of caspase-8 promoter methylation status on TRAIL antitumor activity of human gastric cancer cells and to define the clinical potential of the DNA hypomethylating agent 5-Aza-CdR in human gastric cancer.Methods TRAIL protein was prepared by Ni2+ affinity chromalography method. Three gastric cancer cell lines were treated with 5-Aza-CdR and the antitumor activity of TRAIL was determined by MTT assay. Total RNA was extracted from the treated cells and RT-PCR was used to determine the effect of the treatment on the expression of caspase 8. methylation-specific PCR(MSP) analysis was used to confirm methylation of caspase 8 promoter regions.Results TRAIL showed low antineoplastic activity in gastric cell lines SGc 7901, Kato 3 and AGS. We analysed the methylation status of caspase 8 promoter regions anddemonstrated that CpG island is unmethylated in all three gastric cancer cell lines. Caspase-8 mRNA was expressed, however expression level is low in three cell lines; Exposure to 5-Aza-CdR induced an increased expression of the caspase 8 in the three gastric cancer cells. 5-Aza-CdR also enhanced the antitumor activity of TRAIL in three gastric cancer cells.Conclusions Resistance to TRAIL-induced apoptosis in gastric cancer cells correlates with a loss of caspase-8 expression. Treatment with demethylation agent 5-Aza-CdR can lead to expression of silenced caspase 8 gene and restoration of sensitivity of gastric cancer cells to TRAIL.