K-RAS transformation in prostate epithelial cell overcomes H₂O₂-induced apoptosis via upregulation of gamma-glutamyltransferase-2

Abstract

The anti-apoptotic oncogene K-RAS is hypothesized to increase the antioxidant status of cells, thereby protecting them from generation of reactive oxygen species (ROS). Therefore, we examined whether K-RAS overcomes hydrogen peroxide (H₂O₂)-mediated apoptosis in the human fetal prostate epithelial cell 267B1. In this study, we found that treatment of 267B1 cells with H₂O₂ resulted in significant reduction of cell growth, which was associated with cytochrome-c release and caspase-3 activation. However, mutated K-RAS transformation (268B1/K-RAS) rendered 267B1 cells reduction of the resistance to H₂O₂-induced apoptosis through suppression of ROS generation. In addition, we analyzed profiling of gene expression in K-RAS transformation and found that gamma-glutamyltransferase 2 (GGT2) most highly expressed. Transient knockdown of K-RAS resulted in a significant downregulation of GGT gene expression. We also revealed that expression of GGT2 gene is closely regulated by the ERK signal pathway in 267B1/K-RAS cells. In addition, the anti-apoptotic effect of mutated K-RAS was attenuated by treatment with GGT2 RNA interference through inhibition of ROS generation, suggesting that mutated K-RAS mediates resistance to H₂O₂-induced apoptosis through GGT2 activation. These results importantly provide mechanistic insights on the anti-apoptotic activity of mutated K-RAS.

Introduction

Pancreatic cancer is the fourth common cause of cancer death in the United States and mutated RAS isoforms play a pivotal role in multiple pathways (Jemal et al., 2002). The RAS proto-oncogene superfamily of monomeric membrane-associated GTPases includes the three classic members H-RAS, N-RAS, and K-RAS (Castellano and Santos, 2011, Ellis and Clark, 2000, Hancock, 2003, Plowman et al., 2003). Among them, K-RAS is an anti-apoptotic proto-oncogene that is derived from alternative splicing of exon 4. K-RAS mutations have been identified in up to 95% of pancreatic cancers, implying their important role in the development of pancreatic malignancies (Matthaios et al., 2011, Remmers et al., 2011). Due to its unique stretch of lysines at the COOH terminus, K-RAS is thought to exhibit antioxidant properties that protect cells from hydrogen peroxide (H₂O₂)-mediated cell death (Cuda et al., 2002, Recktenwald et al., 2008). K-RAS is also known to alter antioxidant gene and/or protein expression pattern through activation of many signal transduction pathways (Recktenwald et al., 2008). Although the role of K-RAS in classic signal transduction cascades, such as the ERK signal pathway (Marshall, 1995, Plattner et al., 1999), is well understood, its influence on the expression/activation of proteins involved in gene transcription of antioxidants, has not yet been analyzed in detail. Moreover, it is still unclear how K-RAS regulates antioxidant proteins, thereby protecting cells from H₂O₂-induced cell death.

To investigate the function of K-RAS in H₂O₂-induced cell death, we used the 267B1 and 267B1/K-RAS cells. The human fetal prostate epithelial 267B1 cells, immortalized by the SV40 T antigen, are non-tumorigenic and cannot form colonies in soft agar. On the other hand, the v-K-RAS transformed 267B1/K-RAS cells are tumorigenic. By microarray analyses, we examined the 267B1/K-RAS cells for expression of genes that control antioxidant status by K-RAS activation. Interestingly, the gamma-glutamyltransferase-2 (GGT2) transcript was significantly upregulated in K-RAS-transformed human prostate cells. Using the 267B1 and 267B1/K-RAS cells, we investigated the regulatory mechanism of GGT2 expression and its role in K-RAS-transformed 267B1 prostate epithelial cells treated with H₂O₂. Our results indicate that K-RAS protected cells from H₂O₂-induced cytotoxicity by inducing upregulation of the antioxidant enzyme GGT2 through generation of reactive oxygen species (ROS) and consequently inhibiting apoptosis.