Abstract A79: Expression of argininosuccinate synthetase (ASS) and response to arginine deiminase (ADI) treatment in a series of melanoma cell lines
Authors: Manca Antonella , Sini Cristina , Izzo Francesco , Ascierto Paolo A. , Tatangelo Fabiana , Cossu Antonio , Budroni Mario , Palmieri Giuseppe 
Published: June 2012

Background: Arginine deiminase (ADI), an enzyme that catalize the conversion of arginine to citrulline, has been used in clinical trials for the treatment of tumors which have been shown to be sensitive to arginine deprivation, such as malignant melanoma (MM) and hepatocellular carcinoma (HCC). We here evaluated the effect of ADI treatment on ASS expression in a series of melanoma cell lines.

Methods: Twenty-five melanoma cell lines (6 derived from primary melanomas and 19 from MM metastases) and normal fibroblast cell as control underwent cell proliferation assays in presence or absence of ADI. The percentage of cell proliferation was estimated on day 6 of the ADI treatment by a colorimetric assay; the IC50 value was assessed for each cell line. Western blot analysis on lysates from treated and untreated cells were performed for evaluating the expression levels of both the ASS and the ornithine carbamoyl transferase (OTC) proteins. A subset of tissue sections from primary melanomas (N=10) and hepatocellular carcinomas (N=20) was investigated by immunohistochemistry for ASS expression.

Results: Overall, 21/25 (84%) MM cell lines presented a cell growth inhibition with a median IC50 value of 1.3 mU/mL (range, 0.4–2.0); growth of the remaining 4 (16%) MM cell lines was not inhibited and the IC50 was not reached. As expected, proliferation of the control fibroblasts was not inhibited. Expression analysis revealed that none of the 21 ADI-responsive cell lines presented detectable levels of the ASS protein before the ADI treatment; conversely, the 4 melanoma cell lines and the normal fibroblasts, which did not reached an IC50 value, showed a constitutive ASS expression in both treated and untreated cells. After treatment with ADI at a concentration corresponding to the IC50 values, 7/21 (33%) melanoma cell lines presented a markedly increased expression levels of the ASS protein; interestingly, six of them were from paired primary and secondary lesions of three patients. The median value of IC50 in this subset was of 1.8 mU/mL (range, 1.2–2.0). Nearly all samples (13/14; 93%) with absence of the ASS expression in both treated and untreated cells originated from MM metastases; among them, three cell lines were from paired primary and secondary lesions of the same patient. The median value of IC50 in this subset was of 1.2 mU/mL (range, 0.4–1.7). A constitutive expression of the OTC protein was found in all melanoma cell lines and no change in OTC expression was observed after the ADI treatment. Lack of ASS immunostaining was observed in all analyzed in vivo specimens (10 melanomas and 20 hepatocellular carcinomas).

Conclusions: In our series, the most effective cell growth inhibition was observed in melanoma cells with absence of ASS expression either before or after the ADI treatment.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A79.