title: Down-regulation of argininosuccinate synthetase is associated with cisplatin resistance in hepatocellular carcinoma cell lines: implications for PEGylated arginine deiminase combination therapy.
abstract: BACKGROUND
  Many advanced human tumors, including hepatocellular carcinomas (HCC) are auxotrophic for arginine due to down-regulation of argininosuccinate synthetase (ASS1), the rate-limiting enzyme in arginine synthesis. The arginine-lowering agent PEGylated arginine deiminase (ADI-PEG 20) has shown efficacy as a monotherapy in clinical trials for treating arginine-auxotrophic tumors and is currently being evaluated in combination with cisplatin in other cancer types. Epigenetic silencing via methylation of the ASS1 promoter has been previously demonstrated in other cancer types, and a reciprocal relationship between ASS1 expression and cisplatin resistance has also been observed in ovarian cancer. However, the mechanism of ASS1 down-regulation, as well as the correlation with cisplatin resistance has not been explored in HCC. The present study investigates ADI-PEG 20 and cisplatin sensitivities in relation to ASS1 expression in HCC. In addition, we show how this biomarker is regulated by cisplatin alone and in combination with ADI-PEG 20.
  
  
  METHODS
  ASS1 protein expression in both untreated and drug treated human HCC cell lines was assessed by western blot. The correlation between ASS1 protein levels, ADI-PEG 20 sensitivity and cisplatin resistance in these cell lines was established using a luminescence-based cell viability assay. Epigenetic regulation of ASS1 was analyzed by bisulfite conversion and methylation-specific PCR.
  
  
  RESULTS
  A good correlation between absence of ASS1 protein expression, ASS1 promoter methylation, sensitivity to ADI-PEG 20 and resistance to cisplatin in HCC cell lines was observed. In addition, cisplatin treatment down-regulated ASS1 protein expression in select HCC cell lines. While, at clinically relevant concentrations, the combination of ADI-PEG 20 and cisplatin restored ASS1 protein levels in most of the cell lines studied.
  
  
  CONCLUSIONS
  ASS1 silencing in HCC cell lines is associated with simultaneous cisplatin resistance and ADI-PEG 20 sensitivity which suggests a promising combination therapeutic strategy for the management of HCC.
authors.affiliation: Department of Biology, Polaris Pharmaceuticals, 9373 Towne Center Drive, Suite #150, San Diego, CA 92121, USA. jmcalpine@polarispharma.com.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: McAlpine
authors.forename: Hsin-Tze
authors.initials: HT
authors.lastname: Lu
authors.forename: Katherine C
authors.initials: KC
authors.lastname: Wu
authors.forename: Susan K
authors.initials: SK
authors.lastname: Knowles
authors.forename: James A
authors.initials: JA
authors.lastname: Thomson
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D000971:Antineoplastic Combined Chemotherapy Protocols; D001124:Argininosuccinate Synthase / Q000235:genetics / Q000378:metabolism*; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000378:metabolism*; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000494:pharmacology*; D019175:DNA Methylation; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D004357:Drug Synergism; D056945:Hep G2 Cells; D006801:Humans; D006867:Hydrolases / Q000494:pharmacology*; D008113:Liver Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D011092:Polyethylene Glycols; D006867:Hydrolases; C512527:ADI PEG20; D001124:Argininosuccinate Synthase; D002945:Cisplatin