Background: Many advanced human tumors, including hepatocellular carcinomas (HCC) are auxotrophic for\narginine due to down-regulation of argininosuccinate synthetase (ASS1), the rate-limiting enzyme in arginine\nsynthesis. The arginine-lowering agent PEGylated arginine deiminase (ADI-PEG 20) has shown efficacy as a\nmonotherapy in clinical trials for treating arginine-auxotrophic tumors and is currently being evaluated in\ncombination with cisplatin in other cancer types. Epigenetic silencing via methylation of the ASS1 promoter has\nbeen previously demonstrated in other cancer types, and a reciprocal relationship between ASS1 expression and\ncisplatin resistance has also been observed in ovarian cancer. However, the mechanism of ASS1 down-regulation, as\nwell as the correlation with cisplatin resistance has not been explored in HCC. The present study investigates\nADI-PEG 20 and cisplatin sensitivities in relation to ASS1 expression in HCC. In addition, we show how this\nbiomarker is regulated by cisplatin alone and in combination with ADI-PEG 20.\nMethods: ASS1 protein expression in both untreated and drug treated human HCC cell lines was assessed by\nwestern blot. The correlation between ASS1 protein levels, ADI-PEG 20 sensitivity and cisplatin resistance in these\ncell lines was established using a luminescence-based cell viability assay. Epigenetic regulation of ASS1 was\nanalyzed by bisulfite conversion and methylation-specific PCR.\nResults: A good correlation between absence of ASS1 protein expression, ASS1 promoter methylation, sensitivity\nto ADI-PEG 20 and resistance to cisplatin in HCC cell lines was observed. In addition, cisplatin treatment\ndown-regulated ASS1 protein expression in select HCC cell lines. While, at clinically relevant concentrations, the\ncombination of ADI-PEG 20 and cisplatin restored ASS1 protein levels in most of the cell lines studied.\nConclusion: ASS1 silencing in HCC cell lines is associated with simultaneous cisplatin resistance and ADI-PEG 20\nsensitivity which suggests a promising combination therapeutic strategy for the management of HCC.
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