|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Cancer cell sensitivity to arginine deprivation in vitro is not determined by endogenous levels of arginine metabolic enzymes
Yaroslav P Bobak, Bozhena O Vynnytska, Yuliya V Kurlishchuk, Andriy A Sibirny and Oleh V Stasyk1
Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine
Single amino acid Arg (arginine) deprivation is currently considered as a therapeutic approach to treat certain types of tumours; the molecular mechanisms that underlie tumour cell sensitivity or resistance to Arg restriction are still little understood. Here, we address the question of whether endogenous levels of key Arg metabolic enzymes [catabolic: arginases, ARG1 (arginase type 1) and ARG2 (arginase type 2), and anabolic: OTC (ornithine transcarbamylase) and ASS (argininosuccinate synthetase)] affect cellular responses to arginine deprivation in vitro. Human epithelial cancer cells of different organs of origin exhibiting variable sensitivity to Arg deprivation provided the experimental models. Neither the basal expression status of the analysed enzymes, nor their changes upon arginine withdrawal correlated with cancer cell sensitivity to arginine deprivation. However, the ability to utilize exogenous Arg precursors (ornithine and citrulline) for growth in Arg-deficient medium strongly correlated with expression of the corresponding enzymes, OTC and ASS. We also observed that OTC expression was below the level of detection in all the types of tumour cells analysed, suggesting that in vitro, at least for them, Arg is an essential amino acid.
Key words: arginine deprivation, cell death, urea cycle enzyme
Abbreviations: Arg, arginine, ARG1, arginase type 1, ARG2, arginase type 2, ASS, argininosuccinate synthetase, Cit, citrulline, Orn, ornithine, OTC, ornithine transcarbamylase, PARP, poly (ADP–ribose) polymerase
1To whom correspondence should be addressed (email email@example.com).
Part of a series marking the 70th birthday of the Cell Biology International Editor-in-Chief Denys Wheatley
Arg (arginine) is classified as a semiessential amino acid in humans with multiple metabolic and regulatory functions. Arg is not only required for protein synthesis, but is involved in the urea cycle, metabolism of other amino acids and nucleotides, biosynthesis of nitric oxide, polyamines, agmatine and creatine, which are important regulators of cell growth and survival (Delage et al., 2010).
In the adult human, the diet and intracellular protein degradation ensure most of the Arg requirement at the physiological level (Choi et al., 2009). Besides, Arg can be synthesized de novo from its precursors [Orn (ornithine) and Cit (citrulline)] in sequential reactions of the urea cycle in the so-called ‘intestinal–renal axis’ (Morris, 2006). ASS (argininosuccinate synthetase) is the key rate-limiting enzyme in Arg biosynthesis (Husson et al., 2003). Normal cells can convert Cit (endo- or exogenous) into Arg, while some malignant cells are ASS-deficient and, therefore, fully dependent on an exogenous Arg supply (Dillon et al., 2004; Wheatley et al., 2005). Such metabolic dependence of tumour cells was exploited to develop enzymatic antitumour therapy based on recombinant Arg-degrading enzymes, human arginase I or bacterial arginine deiminase (Delage et al., 2010; Glazer et al., 2010). In animal models as well as in clinical trials, Arg deprivation in vivo is well tolerated, while being detrimental for the progression of tumours, such as hepatocarcinomas (Izzo et al., 2004) and melanomas (Ascierto et al., 2005).
However, because of the complexity of Arg-dependent signalling and metabolic networks, the molecular mechanisms that determine cell sensitivity to Arg starvation need to be fully elucidated. Up to now, it has not been systematically assayed whether intracellular Arg metabolism, its catabolic degradation and its de novo synthesis, affects cell sensitivity to Arg deprivation in vitro. To address this question, we have monitored the expression of key enzymes of Arg metabolism [ARG1 (arginase type 1), ARG2 (arginase type 2), ASS and OTC (ornithine transcarbamylase)] under normal culture conditions and after Arg deprivation in six human epithelial cell lines of different origin, and measured cell survival and the ability to retain growth potential under Arg starvation. Our data demonstrate that, despite being incapable of Arg synthesis de novo, tumour cells exhibit widely varying sensitivity to Arg deprivation.
2. Materials and methods
2.1. Cell lines and cell culture
Cell lines were obtained from different sources – keratinocytic carcinoma A431, lung adenocarcinoma A549, hepatocellular carcinoma HepG2, breast adenocarcinoma MCF7 and embryonic kidney cells HEK293 from the R. E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology (Kyiv, Ukraine) and SK-MEL-28 cells from the Institute of Cell Biology and Immunology, University of Stuttgart (Stuttgart, Germany). All cells were cultured in Dulbecco's modified Eagle's medium (HyClone Laboratories) supplemented with 10% FBS (fetal bovine serum) (PAA Laboratories GmbH) and 50 μg/ml gentamycin (Sigma–Aldrich).
Experimental Arg-containing (0.4 mM; HyClone) and Arg-free media were supplemented with 5% dialysed FBS (HyClone). Cit (HyClone) and Orn (HyClone) were added to the medium to the final concentration of 0.4 mM (i.e. equimolar to that of Arg).
For cell growth and survival analysis, 1×105 cells were seeded in 24-well plates. Triplicate wells were used for each condition. After 12 h, cells were washed with PBS and the medium changed to the experimental one. After the indicated time-points, the numbers of viable and dead cells were determined by the Trypan Blue exclusion test.
Total RNA was isolated from cells by the method of Chomczynski and Sacchi (1987). First-strand cDNA synthesis was performed using First Strand cDNA Synthesis Kit (Fermentas) and an oligo-dT primer according to the manufacturer's instructions. PCR was performed using a High Fidelity PCR Enzyme Mix (Fermentas) with the following primer pairs:
PCR fragments were separated by electrophoresis on 1.5% agarose gel and visualized by ethidium bromide staining. The relative mRNA expression levels were estimated after normalization with β-actin.
2.3. Western blot analysis
Treated and control cells were lysed in extraction buffer containing 10 mM Tris/HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 5 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 5 mM benzamidine, 1 mM PMSF, 10 μg/ml aprotinin and 1 μg/ml pepstatin. Protein concentration in obtained supernatants was determined according to the method of Peterson (1977).
Equal amounts of total protein were separated on 10% SDS/PAGE and transferred to PVDF membrane (Millipore Corp.). Membranes were probed with primary antibodies against ARG1 (Department of Cell Signaling), ASS (BD Transduction Laboratories), OTC (Atlas Antibodies), PARP [poly(ADP-ribose) polymerase] (Cell Signaling Technologies) and β-actin (Sigma–Aldrich) as the loading control. Secondary goat horseradish peroxidase-conjugated anti-rabbit (Cell Signaling Technology) or anti-mouse (Millipore Corp.) antibodies and an ECL detection system (Millipore Corp.) were used to visualize immunoreactive bands.
2.4. Measurement of arginase activity
Arginase activity in the cell lysates was measured essentially as described by Chang et al. (2001). Briefly, cell lysates were added to the standard assay mixture containing 50 mM Tris/HCl (pH 7.5), 10 mM MnCl2 and 50 mM Arg and incubated at 37°C for 90 min. The reaction was stopped by adding trichloroacetic acid. The amount of urea formed was used as an index of arginase activity, which was assessed spectrophotometrically at 490 nm.
2.5. Measurement of OTC activity
OTC enzymatic activity was measured as described by Yoon et al. (2000) with minor modifications. Cell extracts were incubated with the reaction mixture containing 10 mM Tris/HCl (pH 8.5), 5 mM Orn, 15 mM carbamyl phosphate (Sigma–Aldrich) at 37°C for 30 min. The reaction was stopped with a phosphoric acid/sulfuric acid 3:1 mixture. Cit production was analysed after 15 min of incubation with 3% 2,3-butanedione monoxime (Sigma–Aldrich) at 95–100°C in the dark by measuring absorbance at 490 nm.
All experiments were repeated three times. Parameters of interest were compared using Student's t tests.
3. Results and discussion
3.1. Expression of the key genes of Arg metabolism in human epithelial cell lines
RT-PCR analysis of Arg catabolic gene (ARG1 and ARG2, hydrolyse Arg to Orn) expression under normal (Arg-sufficient) culture conditions showed that ARG1 mRNA could be detected in HepG2 hepatocellular carcinoma cells, but not in other cell lines (Figure 1A). Varying levels of ARG2 transcript were detected in all studied cell lines, with the highest in HepG2 and HEK293 cells (Figure 1A).
Analysis of Arg anabolic gene expression in Arg-sufficient conditions showed that A431, HepG2 and MCF7 cells possessed high levels of ASS (converting Cit into argininosuccinate) mRNA transcript (Figure 1A). A549 and HEK293 had lower ASS expression levels, while in SK-MEL-28 cells, only traces of ASS mRNA were detected (Figure 1A). In contrast, OTC (converting Orn into Cit) transcript was detected only in HepG2 cells (Figure 1A).
Western blot analysis of the correspondent proteins showed that none of the tested cell lines harboured ARG1 and OTC proteins (Figure 1B). The lack of ARG1 and OTC proteins in HepG2 cells that produce ARG1 and OTC mRNA transcripts (Figure 1A) suggests negative post-transcriptional regulation of the corresponding gene expression. From biochemical analysis, all tested cells had no OTC and considerable arginase activities (Figure 1C), except for low arginase activity in HepG2, A549 and HEK293 cells that apparently corresponded to the ARG2 enzyme.
We also demonstrated that the level of ASS protein in all cells was variable, but positively correlated with the level of ASS mRNA transcripts (Figure 1B).
Our data suggest that due to the lack of active OTC enzyme, all the cells tested are, in fact, Arg auxotrophs. OTC expression was additionally assayed in other human cell lines, namely keratinocytes HaCaT, cervical carcinoma HeLa, ovarian carcinoma SKOV3, pancreatic carcinoma MIA PaCa-2 and melanomas MeWo, WM115, WM451 and revealed that they all were OTC negative. This confirms a hypothesis proposed by Wheatley (2005) that the lack of OTC expression is a typical feature of human epithelial cells of different organs of origin.
3.2. Effect of Arg deprivation on expression of the genes of Arg metabolism
One can expect that Arg withdrawal may affect expression of its metabolic enzymes. To elucidate this, we compared expression of ASS, OTC, ARG1 and ARG2 in our model epithelial cells cultured in Arg-containing and Arg-free media for 24 h.
For A431 and HepG2 cells, which exhibited high ASS expression under Arg-sufficient conditions, Arg starvation had no significant effect on ASS mRNA level (Figure 2A). For MCF7 cells that also possessed a relatively high level of ASS transcript and HEK293 cells with low ASS expression detected in Arg-sufficient medium, 1.5- and 2-fold increases in ASS mRNA, respectively, were detected in Arg-free medium (Figure 2A). Thus, under Arg-deprived conditions, the expression of ASS in HEK293 was elevated to the level observed in cells with high basal ASS expression (A431, MCF7 and HepG2). For two other cell lines (A549 and SK-MEL-28) with low ASS level in Arg-supplemented medium, no Arg starvation-induced up-regulation of ASS expression was detected (Figure 2A). Corresponding proportionality to ASS mRNA alterations found at the ASS protein level in different cell lines was demonstrated by Western blot analysis (data not shown).
No changes in the expression levels of OTC and ARG2 due to Arg deprivation relative to Arg-supplemented medium were observed in all cell lines following RT-PCR analysis. ARG1 mRNA was decreased 2-fold in HepG2 cells in Arg-free medium and was not detectable in all other cells analysed (data not shown). The data suggest that Arg deficiency can induce significant up-regulation of the ASS (but not OTC, ARG1 or ARG2) expression in some human epithelial cell lines.
3.3. Effect of the Arg anabolic enzymes on utilization of exogenous Arg precursors
To test whether ASS expression level correlates with the ability of cells to utilize Cit as an Arg precursor, we compared cell proliferation after 72 h in Arg-sufficient, Arg-free and Arg-free medium supplemented with Cit (at equimolar to Arg concentration). As expected, Arg starvation abolished proliferation of all studied cell lines. Cit supplementation resulted in growth restoration of A431, MCF7 and HepG2 cells with high basal expression of ASS, and of HEK293 cells for which induction of ASS expression upon Arg starvation was detected (Figure 2B). A549 cells, with low ASS expression level both under Arg-sufficient and Arg-deprived conditions, were only capable of weak proliferation upon supplementation with Cit (Figure 2B). For SK-MEL-28 cells with barely detectable ASS transcript under all tested conditions, no growth on Cit was observed (Figure 2B).
Thus, ASS expression positively correlates with the cellular ability to utilize Cit as a growth substrate in Arg-free medium, also implying intact ASS and argininosuccinate lyase enzymatic activities in the studied cells. OTC deficiency prevents utilization of Orn as an Arg substitute.
3.4. Capacity for Arg de novo synthesis does not determine tumour cell sensitivity to Arg deprivation in vitro
We measured sensitivity to Arg starvation in three model cell lines (A549, A431 and HepG2) that all lacked OTC activity and differed in the ASS expression level (Figure 1). For this purpose, cell death dynamics following Arg starvation, fragmentation of the reparation enzyme PARP (as an indicator of apoptosis), as well as cell ability to recover proliferation after Arg starvation, were tested.
Cell viability analysis showed that the number of dead A549 cells did not increase during Arg deprivation, whereas it increased time-dependently in the A431 and HepG2 cell lines (Figure 3A). To test whether Arg deprivation-induced death was attributable to apoptosis, we investigated PARP status in the corresponding cell lines after 72 h of Arg starvation. In A431 and HepG2 cells, Arg deprivation induced massive PARP degradation, whereas in A549 cells, no PARP fragmentation and/or degradation could be detected (Figure 3B). These data were also in accordance with results of DNA fragmentation assay (data not shown). We also observed that apoptotic manifestation negatively correlated with the cell ability to retain growth potential upon Arg starvation. Thus, A549 cell capacity to restore growth did not change over the starvation time-course, whereas for A431 and HepG2 cells, it decreased time-dependently (Figure 3C).
Our data demonstrate that despite being OTC and (partially) ASS deficient and, as a result, incapable of intracellular Arg de novo synthesis, A549 cells exhibit high resistance to Arg deprivation in vitro. On the other hand, HepG2 and A431 cells (OTC deficient but ASS positive) are very sensitive to Arg deprivation in vitro and die by apoptosis. No apparent correlation between cell sensitivity to Arg deprivation and intracellular arginase activity was observed.
In summary, the results of this study imply that neither deficient Arg anabolism leads to, nor intracellular arginase activity affects, tumour cell sensitivity to Arg deprivation in vitro. We propose that in vivo ASS deficiency may be required (to prevent exogenous Cit to Arg intracellular conversion), but not sufficient to make certain tumour cells (i.e. lung adenocarcinoma A549 cells) susceptible to enzymatic Arg deprivation. In our next experiments, we plan to identify regulatory mechanism(s) implicated in determining the fate of cell in response to Arg withdrawal in vitro and whether a drop in intracellular free Arg pool is their main trigger.
Study was conceived by Yaroslav Bobak, Oleh Stasyk and Andriy Sybirny. Yaroslav Bobak, Bozhena Vynnytska and Yuliya Kurlishchuk were responsible for data acquisition. The study supervision was performed by Oleh Stasyk. Manuscript revision and final version approval was made by all authors.
The paper is dedicated to the 70th birthday of Professor Denys Wheatley. Authors are thankful to him for inspiring their work and discussing several aspects of this paper.
This work was partly supported by the
Ascierto, PA, Scala, S, Castello, G, Daponte, A, Simeone, E and Ottaiano, A (2005) Pegylated arginine deiminase treatment of patients with metastatic melanoma: results from phase I and II studies. J Clin Oncol 23, 7660-8
Choi, BS, Martinez-Falero, IC, Corset, C, Munder, M, Modolell, M and Muller, I (2009) Differential impact of L-arginine deprivation on the activation and effector functions of T cells and macrophages. J Leukocyte Biol 85, 268-77
Delage, B, Fennell, DA, Nicholson, L, McNeish, I, Lemoine, NR and Crook, T (2010) Arginine deprivation and argininosuccinate synthetase expression in the treatment of cancer. Int J Cancer 126, 2762-72
Dillon, BJ, Prieto, VG, Curley, SA, Ensor, CM, Holtsberg, FW and Bomalaski, JS (2004) Incidence and distribution of argininosuccinate synthetase deficiency in human cancers: a method for identifying cancers sensitive to arginine deprivation. Cancer 100, 826-33
Glazer, ES, Piccirillo, M, Albino, V, Di Giacomo, R, Palaia, R and Mastro, AA (2010) Phase II study of pegylated arginine deiminase for nonresectable and metastatic hepatocellular carcinoma. J Clin Oncol 28, 2220-6
Husson, A, Brasse-Lagnel, C, Fairand, A, Renouf, S and Lavoinne, A (2003) Argininosuccinate synthetase from the urea cycle to the citrulline-NO cycle. Eur J Biochem 270, 1887-99
Izzo, F, Marra, P, Beneduce, G, Castello, G, Vallone, P and De Rosa, V (2004) Pegylated arginine deiminase treatment of patients with unresectable hepatocellular carcinoma: results from phase I/II studies. J Clin Oncol 22, 1815-22
Wheatley, DN (2005) Arginine deprivation and metabolomics: important aspects of intermediary metabolism in relation to the differential sensitivity of normal and tumour cells. Semin Cancer Biol 15, 247-53
Wheatley, DN, Kilfeather, R, Stitt, A and Campbell, E (2005) Integrity and stability of the citrulline–arginine pathway in normal and tumour cell lines. Cancer Lett 227, 141-52
Yoon, JH, Lee, HS, Kim, TH, Woo, GH and Kim, CY (2000) Augmentation of urea-synthetic capacity by inhibition of nitric oxide synthesis in butyrate-induced differentiated human hepatocytes. FEBS Lett 474, 175-8
Received 16 June 2010; accepted 23 July 2010
Published as Cell Biology International Immediate Publication 23 July 2010, doi:10.1042/CBI20100451
© The Author(s) Journal compilation © 2010 Portland Press Limited
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)
Figure 1 Expression analysis of the key genes of Arg metabolism (ARG1, ARG2, OTC, ASS) in human epithelial cell lines