Inducible antisense RNA targeting amino acid transporter ATB0/ASCT2 elicits apoptosis in human hepatoma cells

Bryan C. Fuchs, J. Christian Perez, Julie E. Suetterlin, Sofia B. Chaudhry, Barrie P. Bode
2004 American Journal of Physiology - Gastrointestinal and Liver Physiology  
Inducible antisense RNA targeting amino acid transporter ATB 0 /ASCT2 elicits apoptosis in human hepatoma cells. Amino acid transporter B 0 /ASC transporter 2 (ATB 0 /ASCT2) is responsible for most glutamine uptake in human hepatoma cells. Because this transporter is not expressed in normal hepatocytes, we hypothesized that its expression is necessary for growth of human liver cancer cells. To test this hypothesis, Sloan Kettering hepatoma (SK-Hep) cells were stably transfected with an
more » ... ed with an inducible 1.3-kb ATB 0 /ASCT2 antisense RNA expression plasmid under the transcriptional control of mifepristone, a synthetic steroid. Induced antisense RNA expression in monolayer cultures decreased ATB 0 /ASCT2 mRNA levels by 73% and glutamine transport rates by 65% compared with controls after 24 h, leading to a 98% decrease in cell number after 48 h. Cellular death was attributable to apoptosis based on cellular blebbing, caspase-3 activation, vital dye and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, and poly-(ADP-ribose) polymerase (PARP) cleavage. Transporter knockdown also markedly increased activities of caspases-2 and -9, marginally enhanced caspase-8 activity, and dramatically increased ASCT1 mRNA levels, presumably as a futile compensatory response. Apoptosis elicited via transporter silencing was not attributable to the double-stranded RNA-dependent protein kinase R (PKR) pathway. For comparison, glutamine deprivation also caused apoptotic cell death but with slower temporal kinetics, stimulated caspases-2 and -3 but not caspases-8 or -9 activities, and led to considerable PARP cleavage. Thus ASCT2 suppression exerts proapoptotic effects transcending those of glutamine starvation alone. We conclude that ATB 0 /ASCT2 expression is necessary for SK-Hep cell growth and viability and suggest that it be further explored as a selective target for human hepatocellular carcinoma. caspase; glutamine; hepatocellular carcinoma THE AMINO ACID GLUTAMINE IS avidly utilized by multiple cell types in the body as a metabolic intermediate, contributing carbon and nitrogen for the synthesis of other amino acids, fatty acids, nucleic acids, and proteins, and it also serves as an oxidizable substrate for ATP production in dividing cells (22) . Its pivotal role in cellular metabolism is underscored by its relatively high turnover rate in all cell types (12) and by the fact that it is the most abundant amino acid in plasma at levels around 0.6 mM. Glutamine also serves as the primary nontoxic shuttle for ammonia from extrahepatic tissues to the liver, where it is taken up by the amino acid transporter System N (20) and utilized in the urea cycle, transamination reactions, and gluconeogenesis (27) . On the basis of the unique metabolic heterogeneity along the liver acinus, the liver can rapidly alter flux through glutamine-producing or -consuming pathways and thus serves as a systemic integrating center for glutamine homeostasis (15). In hepatocellular carcinoma (HCC), net glutamine consumption is observed with utilization of this nitrogen-rich amino acid for growth-related pathways (10). Consequently, the systemic glutamine homeostatic function of the liver is subverted, resulting in significantly decreased plasma glutamine levels in patients with liver cancer (16) , an event that can adversely impact glutamine-dependent cells, such as those in the immune system (1). Previous work from our laboratory (6-9) showed that human hepatoma cells take up glutamine at rates 10-to 30-fold faster than normal human hepatocytes via a transporter historically termed System ASC. This high-affinity Na ϩ -dependent glutamine transport activity is not expressed in normal hepatocytes (7). The system ASC transporter was later cloned and termed amino acid transporter B 0 (ATB 0 ) (19) and seems to be the human ortholog of rodent ASC transporter 2 (ASCT2) (5). This transporter has also been identified as a cell surface receptor for mammalian retroviruses and is referred to as human ASCT2 (35). ATB 0 /ASCT2 expression was recently reported to be absent in normal human hepatocytes but present in six human hepatoma cell lines and primary liver tumors and shown to mediate the glutamine-dependent growth of rapidly growing human hepatoma cells (6). These results were confirmed by a recent query at the Cancer Genome Anatomy Project website (http://cgap.nci.nih.gov) that showed no ATB 0 / ASCT2 (accession #U53347) expression in normal human liver [0/34,164 expressed sequence tags (ESTs)] but appreciable expression in liver cancer (17/40,737 ESTs) using the Virtual Northern program. Thus expression of this transporter is activated in human liver cancer. According to the American Liver Foundation, HCC is the most common primary malignant tumor of the liver. HCC is also the leading cause of cancer death in Africa and the developing world (28), and its prevalence in Europe and the United States is on the rise due to increased incidence of viral hepatitis. Currently, there is no effective treatment for HCC other than resection or transplant, and both modalities are often unsuccessful. Given the glutamine reliance of aggressively growing hepatoma cells, we hypothesized that inhibition of ATB 0 /ASCT2 expression would arrest the growth of human liver cancers and might eventually be used as a selective HCC therapy. Here, we utilize the Sloan Kettering hepatoma-1 (SK-Hep1) cell line (13) as a model of an aggressively growing human HCC for three reasons. First, it exhibits the fastest growth rate
doi:10.1152/ajpgi.00344.2003 pmid:14563674 fatcat:blbazoxfdvawfivyvkqmo7ktsu