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. 2008 Oct 15;123(8):1950-5.
doi: 10.1002/ijc.23723.

Pancreatic cancer cell lines deficient in argininosuccinate synthetase are sensitive to arginine deprivation by arginine deiminase

Tawnya L Bowles  1 , Randie Kim, Joseph Galante, Colin M Parsons, Subbulakshmi Virudachalam, Hsing-Jien Kung, Richard J Bold
Affiliations

Pancreatic cancer cell lines deficient in argininosuccinate synthetase are sensitive to arginine deprivation by arginine deiminase

Tawnya L Bowles et al. Int J Cancer. .

Abstract

Eukaryotic cells can synthesize the non-essential amino acid arginine from aspartate and citrulline using the enzyme argininosuccinate synthetase (ASS). It has been observed that ASS is underexpressed in various types of cancers ASS, for which arginine become auxotrophic. Arginine deiminase (ADI) is a prokaryotic enzyme that metabolizes arginine to citrulline and has been found to inhibit melanoma and hepatoma cancer cells deficient of ASS. We tested the hypothesis that pancreatic cancers have low ASS expression and therefore arginine deprivation by ADI will inhibit cell growth. ASS expression was examined in 47 malignant and 20 non-neoplastic pancreatic tissues as well as a panel of human pancreatic cancer cell lines. Arginine deprivation was achieved by treatment with a recombinant form of ADI formulated with polyethylene glycol (PEG-ADI). Effects on caspase activation, cell growth and cell death were examined. Furthermore, the effect of PEG-ADI on the in vivo growth of pancreatic xenografts was examined. Eighty-seven percent of the tumors lacked ASS expression; 5 of 7 cell lines similarly lacked ASS expression. PEG-ADI specifically inhibited growth of those cell lines lacking ASS. PEG-ADI treatment induced caspase activation and induction of apoptosis. PEG-ADI was well tolerated in mice despite complete elimination of plasma arginine; tumor growth was inhibited by approximately 50%. Reduced expression of ASS occurs in pancreatic cancer and predicts sensitivity to arginine deprivation achieved by PEG-ADI treatment. Therefore, these findings suggest that arginine deprivation by ADI could provide a beneficial strategy for the treatment of pancreatic cancer, a malignancy in which new therapy is desperately needed.

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Figures

Figure 1
Figure 1
Immunohistochemical analysis of ASS expression in human pancreatic tumors. (a) pancreatic adenocarcinoma lacking ASS expression (solid arrow), (b) pancreatic adenocarcinoma expressing ASS, (c) benign pancreatic ductal cells expressing ASS, (d) metastatic pancreatic adenocarcinoma lacking ASS expression (dotted arrow) with adjacent hepatocytes demonstrating ASS expression (solid arrow). In panels a–c, only neoplastic glandular tissue of pancreatic adenocarcinoma is observed within a dense field of fibroblasts typical of the dense desmoplastic reaction and chronic pancreatitis of pancreatic adenocarcinoma.
Figure 2
Figure 2
Expression of argininosuccinate synthetase (ASS) in pancreatic cancer cell lines. (a) RT-PCR for ASS mRNA (b) Immunoblotting for ASS protein (c) PCR for ASS DNA, (d) Effect of PEG-ADI on ASS mRNA and protein expression in pancreatic cancer cell lines; RT-PCR for ASS mRNA expression in the absence (−) or presence (+) of PEG-ADI(0.3 μg/mL; 5 days), and (e) Western blotting for ASS protein in the absence (−) or presence (+) of PEG-ADI(0.3 μg/mL; 5 days). Actin is shown to demonstrate loading equivalency.
Figure 3
Figure 3
Cytoxicity and cellular effects of arginine deprivation by ADI in human pancreatic cancer cell lines. (a) Dose response of PEG-ADI (in μg/mL) in MIA-PaCa-2 cells (solid line) and L3.3 cells (dotted line) determined by MTT assay (as described in the Materials and Methods), (b) Cell growth curve of L3.3 cells treated with PEG-ADI (0.3 μg/mL), (c) Cell growth curve of MIA-PaCa-2 cells treated with PEG-ADI (0.3 μg/mL), (d) Cell number of L3.3, MIA-PaCa-2 and PANC-1 after treatment with PBS compared to PEG-ADI (0.3 μg/mL) (* = p < 0.05 vs. control), (e) Induction of apoptosis determined by quantitation of the percentage of cells in the sub-G0 fraction on FACS analysis after 4 days of PEG-ADI (0.3 μg/mL) in L3.3, MIA-PaCa-2 and PANC-1 cells, (f) Quantitation of active caspase-3 by ELISA in MIA-PaCa-2 and L3.3 after 24 hr of PEG-ADI (0.3 μg/mL).
Figure 4
Figure 4
Effect of PEG-ADI on MIA-PaCa-2 xenografts and plasma arginine levels. (a) Growth of established MIA-PaCa-2 xenografts in nude mice after treatment with PBS (Control) or PEG-ADI (5 IU) administered biweekly by intraperitoneal injections, (b) HPLC for plasma amino acid levels 24 hr after PBS or PEG-ADI (5 IU) with notation for location of arginine elution at 110.767 min (note that the entire HPLC data including peaks for arginine metabolites including citrulline has been cropped out), and (c) plasma arginine and related precursors or metabolites from 3 mice 24 hr after PBS or PEG-ADI (5 IU). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
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