Inducible antisense RNA targeting amino acid transporter ATB0/ASCT2 elicits apoptosis in human hepatoma cells
Inducible antisense RNA targeting amino acid transporter ATB0/ASCT2 elicits apoptosis in human hepatoma cells
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DOI：
10.1152/ajpgi.00344.2003
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发表时间：
2004-03-01
影响因子：
4.5
通讯作者：
Bode, BP
中科院分区：
文献类型：
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作者：
Fuchs, BC;Perez, JC;Bode, BP
氨基酸转运蛋白B-0/ASC转运蛋白2（ATB（0）/ASCT 2）负责人肝癌细胞中的大多数谷氨酰胺摄取。由于这种转运蛋白在正常肝细胞中不表达，我们假设它的表达对于人类肝癌细胞的生长是必要的。为了验证这一假设，在米非司酮（一种合成类固醇）的转录控制下，用可诱导的1.3-kb ATB（0）/ASCT 2反义RNA表达质粒稳定转染Sloan Kettering肝癌（SK-Hep）细胞。与对照组相比，24 h后单层培养物中诱导的反义RNA表达使ATB（0）/ASCT 2 mRNA水平降低73%，谷氨酰胺转运率降低65%，导致48 h后细胞数量减少98%。细胞死亡可归因于基于细胞起泡、半胱天冬酶-3活化、活体染料和末端脱氧核苷酸转移酶介导的dUTP缺口末端标记染色以及聚（ADP-核糖）聚合酶（PARP）裂解的细胞凋亡。转运蛋白敲除还显著增加了caspase-2和-9的活性，略微增强了caspase-8的活性，并显著增加了ASCT 1 mRNA水平，可能是一种无效的代偿反应。通过转运蛋白沉默引起的细胞凋亡不是由双链RNA依赖的蛋白激酶R（PKR）途径引起的。相比之下，谷氨酰胺剥夺也引起凋亡性细胞死亡，但时间动力学较慢，刺激半胱天冬酶-2和-3但不刺激半胱天冬酶-8或-9活性，并导致相当大的PARP裂解。因此，ASCT 2抑制发挥的促凋亡作用超越了单独的谷氨酰胺饥饿。我们的结论是，ATB 0/ASCT 2的表达是必要的SK-Hep细胞的生长和活力，并建议进一步探讨作为一个选择性靶向人肝细胞癌。
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 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 ATB0/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.