Arginine deprivation as a targeted therapy for cancer

Abstract

Certain cancers may be auxotrophic for a particular amino acid, and amino acid deprivation is one method to treat these tumors. Arginine deprivation is a novel approach to target tumors which lack argininosuccinate synthetase (ASS) expression. ASS is a key enzyme which converts citrulline to arginine. Tumors which usually do not express ASS include melanoma, hepatocellular carcinoma, some mesotheliomas and some renal cell cancers. Arginine can be degraded by several enzymes including arginine deiminase (ADI). Although ADI is a microbial enzyme from mycoplasma, it has high affinity to arginine and catalyzes arginine to citrulline and ammonia. Citrulline can be recycled back to arginine in normal cells which express ASS, whereas ASS(-) tumor cells cannot. A pegylated form of ADI (ADI-PEG20) has been formulated and has shown in vitro and in vivo activity against melanoma and hepatocellular carcinoma. ADI-PEG20 induces apoptosis in melanoma cell lines. However, arginine deprivation can also induce ASS expression in certain melanoma cell lines which can lead to in vitro drug resistance. Phase I and II clinical trials with ADI-PEG20 have been conducted in patients with melanoma and hepatocellular carcinoma, and antitumor activity has been demonstrated in both cancers. This article reviews our laboratory and clinical experience as well as that from others with ADI-PEG20 as an antineoplastic agent. Future direction in utilizing this agent is also discussed.

Fig. (1)

Enzymes involved in urea cycle. The dot line on ADI designate its action on extracellular conversion of arginine to citrulline and ammonia.

Fig. (2)

In situ end labeling apoptosis assay in A375 cell line. A: control. B: after exposure to 0.08 ug/ml for 72 hr. Treated cells undergo apopotosis shown brown staining in the nuclei

Fig. (3)

Apoptosis as detected by PARP cleavage in 4 melanoma cell lines (A375, Sk-mel-2, A2058 and MEL-1220). Untreated cell showed uncleaved PARP at 116 kD whereas treated cells showed cleaved PARP seen at 89 kD.

Fig. (4)

A). The activity of mTOR was measured by its ability to phosphorylated 4E-BP. The bottom band is the unphosphorylated form. 4E-BP. 4E-BP has multiple phosphrylation which depict multiple bands seen. After treatment with ADI-PEG at 0.6 ug/ml (81 IU/ml=11.3 mg/ml) for 72 hr., the major form of 4E-BP are unphosphorylated. B). phosphoryltion of p-70S6 kinase also reflect mTOR activity. After exposure to ADI-PEG20 at 0.6 mg/ml, the phospho-p-70S6 kinase decresaed in A-375, Sk-Mel2, but increase in A-2058, which indicative of phosphorylation from other upstream protein. Mel-1220 has minimal phosphorylation of p-70S6 kinase and hence changes is not discernable. C). phospho-AMPK in 4 melanoma cell lines after exposure to ADI-PEG20 for 72 ht. Three cell lines has increased phospho-AMPK, but no changes in Sk-Mel 2.

Fig. (5)

Southernblot Analysis of ASS in 4 melanoma cell lines and BJ-1 cell lines. All five cell lines show similar levels of ASS DNA.

Fig (6)

Immunoblot of ASS protein in 4 melanoma cell lines (A375, Sk-mel-2, A2058 and MEL-1220) before and after exposure arginine free media supplemented with citrulline and NH4Cl for 24, 48 and 72 hr. Afterward, cells were washed, and replenished with normal EMEM media for 24, 48 and 72 hr. Lane 1: control. Lane 2: 24 hrs. on arginine free media Lane 3: 48 hrs on arginine free media Lane 4: 72 hrs on arginine free media Lane 5: Removal of arginine free media and changed to normal EMEM media for 24 hrs. Lane 6: 48 hrs. on normal media. Lane 7: 72 hrs. on normal media. Similar results were obtained with arginine free media with no citrulline supplement and ADI-PEG20 treated media. with citrulline and NH4Cl supplement.

Fig. (7)

Northernblot Analysis of ASS in a panel of melanoma cell line and BJ-1 cells. Lane 1: A375. Lane 2: A375 after exposure to ADI-PEG20 for 72 hrs. Lane 3: SK-Mel-2 Lane 4: Sk-Mel-2 after exposure to ADI-PEG20 for 72 hrs. Lane 5: MEL-1220. Lane 6: MEL-1220 after exposure to ADI-PEG20 for 72 hrs. Lane 7: A2058. Lane 8: A2058 after exposure to ADI-PEG20 for 72 hrs. Lane 9: A2058R. Lane 10: BJ-1. Lane 11: BJ-1 after exposure to ADI-PEG20 for 72 hrs. Note: only A2058R and BJ-1 possess 1.9 kb ASS mRNA and there is no differences in ASS mRNA in BJ-1 cells after exposure to ADI-PEG20.

Fig (8)

The relative expression level of ASS mRNA detected with real-time PCR in different cell lines in arginine-free (− Arg) media as compared with that in normal media (+Arg). The quantification method used was proposed by Pfaffl in Nucleic Acid Research, 29:2002–2007. GAPDH was used as the reference gene. The hours on the graph indicates the time for which the cells had been cultured in −Arg or +Arg media.

Fig. (9)

Immunoblot of ASS protein in A-2058R (resistance to ADI) after culture in normal media for 7 and 29 days. The expression is stable with no changes.