Deprivation of arginine by recombinant human arginase in prostate cancer cells

Quantitative real-time PCR was performed in prostate cancer cells to detect mRNA expression of ASS, OCT, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data were processed and presented with cycle threshold (Ct) value of each quantitated expression as listed in Table 1. Housekeeping gene GAPDH was used as a reference gene for quantitative expression analysis. The Ct is defined as the number of cycles required for the fluorescent signal to cross the threshold level. Ct is a relative measure of the target mRNA in the PCR, and inversely proportional to the amount of target mRNA. Ct value of 40 or higher means no amplification due to absent gene expression. Abundant expression of ASS mRNA was detected in all three cell lines. Expression of OCT mRNA was absent in LNCaP, and minimally detected in DU-145 and PC-3.

We further determined cell viability after 72-h exposure to rhArg at 0, 0.001, 0.01, 0.1 and 0.5 U/ml. All 3 prostate cancer cell lines were very sensitive to arginine depletion with half maximal inhibitory concentration (IC₅₀) of rhArg less than or equal to 0.02 U/ml. The IC₅₀ of rhArg in these 3 prostate cancer cell lines was similar to the reported values in melanoma and hepatoma cell lines lacking OCT activity [11, 13]. We further investigated the function of OCT by incubating cells with 0.1 U/ml of rhArg (>IC₅₀) in the absence and presence of increasing concentrations of ornithine for 72 h. We expected ornithine could rescue rhArg-mediated cytotoxicity if OCT was expressed and functional. As shown in Figure 1, supplement of ornithine failed to rescue cytotoxicity from rhArg in all 3 cell lines. These results were in consistent with deficient OCT gene expression as demonstrated by quantitative real-time PCR.

Autophagy is mediated by lysosomal degradation, and is an alternative process of cell death [21]. Autophagy is induced by inhibition of mTOR, which is a key sensor and regulator of growth signal and environmental stress [22]. Deprivation of arginine inhibits mTOR pathway and dephosphorylates downstream targets such as 4E-BP1 in Chinese hamster ovary and human melanoma cells [17, 23]. To elucidate the mTOR signaling following arginine depletion by rhArg, we investigated the phosphorylation pattern of 4E-BP1. Since our preliminary result showed citrulline could partially reverse the cytotoxicity from rhArg, cells were treated with either citrulline, rhArg, or both for 48 h, followed by Western blot analysis (Figure 2). Decreased phosphorylation of 4E-BP1 in PC-3 and DU-145 was noted upon exposure to rhArg, irrespective of citrulline supplement. However, phosphorylation pattern of 4E-BP1 did not change in LNCaP. Though inhibition of mTOR by rhArg was noted in PC-3 and DU-145, the partial reversal of cytoxicity by citrulline might not be related to mTOR signaling since we did not observe any difference in phosphorylation pattern of 4E-BP1 in the presence or absence of citrulline.

Apoptosis was determined by DNA fragmentation using TUNEL (terminal deoxynucleotide transferase dUTP nick end labeling) assay after 36-h co-culture of prostate cancer cells with rhArg. Purple, green, pink, blue, and orange histograms indicate levels of DNA fragmentation upon exposure with 0, 0.001, 0.01, 0.1, and 0.5 U/ml rhArg, respectively (Figure 3). The results demonstrated no induction of apoptosis after 36-h exposure of rhArg.

Deficiency in ASS expression renders cellular sensitivity against ADI-PEG20 in prostate cancer [18]. Both LNCaP and PC-3 have been shown to express ASS, and are resistant to arginine depletion by ADI-PEG20 [18]. In our study, all 3 prostate cancer cell lines including LNCaP and PC-3 expressed ASS but had either minimal or absent expression of OCT, and all 3 lines were highly susceptible to arginine deprivation by rhArg. Moreover, sensitivity to rhArg treatment was independent of hormone sensitivity and not affected by ASS expression in our study.

In human melanoma and prostate cancer cells with down-regulated ASS expression, treatment of ADI-PEG20 activates adenosine 5′-monophosphate-activated protein kinase (AMPK) due to decreased ATP levels upon arginine deprivation [18, 24]. Activated AMPK further inhibits mTOR signaling by reducing phosphorylation of 4E-BP1, and leads to autophagy which is a cellular self-degrading process mediated by lysosomes. Kim et al. have shown arginine deprivation by ADI-PEG20 immediately activated AMPK, and formed intense autophagosome in CWR22Rv1 prostate cancer cells within 90 min of ADI-PEG20 exposure [18]. Onset of caspase-independent apoptosis in ~30% CWR22Rv1 cells did not occur until after 96-h exposure of ADI-PEG20. Similar findings of delayed-onset but caspase-dependent apoptosis after arginine deprivation with 3 to 6 days exposure of either ADI-PEG20 or rhArg were reported by different groups [13, 24].

Common stimuli can induce autophagy and apoptosis, which occur either in combined manner or sequential event [25]. It is unclear about the functional relationship between autophagy and apoptosis upon arginine deprivation with either ADI-PEG20 or rhArg. It is possible that upon initial arginine deprivation, autophagy is activated as a defense mechanism to suppress caspase-dependent apoptosis. As arginine deprivation persists more than 72 h, autophagy may give in to caspase-dependent apoptosis in some cell types; whereas in certain cancer cells, autophagy lasting longer than 24 h may lead to caspase-independent form of programmed cell death (autophagic type II cell death) [26].

Using culture media deficient in L-arginine, Wheatley et al. studied the effects of arginine deprivation in 26 cancer cell lines, including PC-3 [27]. They demonstrated clear evidence of cell death during second day of arginine deprivation, and most of PC-3 cells died within 3 days. Furthermore, they observed significantly increased phagosome/lysosome activity from 24 to 36 h after arginine deprivation, suggestive of lytic cell death such as autophagy rather than apoptosis. In this study, we did not identify any significant apoptosis induction after 36-h exposure of rhArg in all 3 cell lines. Additionally, inhibition of mTOR signaling manifested by decreased phosphorylation of 4E-BP1 was noted in DU-145 and PC-3 cells after 48-h exposure of rhArg. Our results are consistent with the report from Wheatley and others, indicating cell death by arginine deprivation in DU-145 and PC-3 is due to autophagic cell death.

Both rhArg and ADI are developed for arginine deprivation in cancer treatment, and currently undergoing clinical investigation. rhArg exhibits significant cytotoxicity against cancer cells such as prostate cancer, melanoma, and hepatocellular carcinoma with OCT deficiency. ADI is effective in tumor cells lacking ASS. Therefore, cancer can be ADI-resistant but rhArg-sensitive, and vice versa. Personalized medicine can be achieved by examining the expression of OCT and ASS in cancer specimen before subjecting cancer patients to arginine deprivation therapy.

DU-145, LNCaP and PC-3 human prostate cancer cells were obtained from the American Type Culture Collection (Manassas, VA). DU-145 and PC-3 are androgen-independent, and LNCaP is androgen-dependent [28]. Cell lines were maintained in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum and antibiotics at 37°C in a humidified atmosphere of 5% CO₂. rhArg was kindly provided by Bio-Cancer Treatments International Ltd. (Hong Kong, China), and was characterized as described previously [11].

Total RNA was extracted using TRIzol reagent (Life Technologies), and cDNA was transcribed from total RNA using SuperScript II RT kit (Life Technologies). Quantitative real-time PCR was performed in triplicate on a 7300 Real Time PCR System, using Gene Expression Assays for ASS, OCT, and GAPDH genes (Applied Biosystems, Foster City, CA). Data were processed and presented with Ct value of each gene expression.

Cells were plated at 10⁴ cells per well in a 96-well plate with increasing concentrations of rhArg at 0, 0.001, 0.01, 0.1 and 0.5 U/ml for 72 h at 37⁰ C. Subsequently, cell viability was determined by a colorimetric method using CellTiter 96 Aqueous Non-radioactive Cell Proliferation Assay according to the manufacturer’s protocol (Promega, Madison,WI).

Protein extraction and Western blot analysis were carried out as previously described with some modifications [29]. After treatment, cells were washed twice with cold phosphate-buffered saline, and then resuspended in lysis buffer (phosphate-buffered saline containing 1% Nonidet P-40, 0.5% sodium deoxycholate and 0.1% SDS) containing the protease inhibitors (100 μg/ml phenylmethylsulfonyl fluoride, 25 μg/ml aprotinin, 25 μg/ml leupeptin, 10 μg/ml soybean trypsin inhibitor and 1 mM sodium orthovanadate). The lysate was incubated on ice for 30 min, passed through a 21 gauge needle twice, and then centrifuged at 15,000 x g for 20 min at 4°C. Protein concentration was determined using the Bio-Rad protein assay. Whole cell lysate containing 50 μg of protein from each sample were used in immunoblotting, and subsequently the gels were electroblotted onto PVDF membranes (Immobilon-P, Millipore). Antibodies purchased from Cell Signaling Technology (Danvers, MA) were used to detect the proteins of interest. The horseradish peroxidase conjugated antibodies against mouse, rabbit and goat IgG were used as secondary antibodies (Sigma-Aldrich, St. Louis, MO). The secondary antibody binding was detected by ECL Plus chemiluminescent reagents and analyzed by Storm image analysis systems (GE Healthcare Biosciences, Piscataway, NJ).

Apoptosis was determined by DNA fragmentation using ApoDirect TUNEL assay kit from Millipore (Billerica, MA) based on supplier’s instruction. Briefly, 10⁶ Cells were incubated with increasing concentrations of rhArg for 36 h. Afterwards, DNA breaks were fluorescently labeled with fluorescein isothiocyanate, and cells were analyzed by FACScan flow cytometer (Becton Dickinson Biosciences, San Jose, CA) using Cell Quest Pro software.

All experiments have been performed at least twice with similar results, and the results of one representative experiment are reported. Cell viability results are reported as the average of 3 experiments with error bars representing standard error of the mean as shown in Figure 1.