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. Author manuscript; available in PMC: 2017 Aug 4.
Published in final edited form as: Mol Pharm. 2015 Dec 17;13(1):262–271. doi: 10.1021/acs.molpharmaceut.5b00706

Selective intracellular delivery of recombinant arginine deiminase (ADI) using pH-sensitive cell penetrating peptides to overcome ADI resistance in hypoxic breast cancer cells

Tzyy-Harn Yeh a,b,‡, Yun-Ru Chen c,‡, Szu-Ying Chen c,d, Wei-Chiang Shen b, David K Ann c,*, Jennica L Zaro b,*, Li-Jiuan Shen a,e,f,*
PMCID: PMC5544535  NIHMSID: NIHMS768214  PMID: 26642391

Abstract

Arginine depletion strategies, such as pegylated recombinant arginine deiminase (ADI-PEG20), offer a promising anticancer treatment. Many tumor cells have suppressed expression of a key enzyme, argininosuccinate synthetase 1 (ASS1), which converts citrulline to arginine. These tumor cells become arginine auxotrophic, as they can no longer synthesize endogenous arginine intracellularly from citrulline, and are therefore sensitive to arginine depletion therapy. However, since ADI-PEG20 only depletes extracellular arginine due to low internalization, ASS1-expressing cells are not susceptible to treatment since they can synthesize arginine intracellularly. Recent studies have found that several factors influence ASS1 expression. In this study, we evaluated the effect of hypoxia, frequently encountered in many solid tumors, on ASS1 expression and its relationship to ADI-resistance in human MDA-MB-231 breast cancer cells. It was found that MDA-MB-231 cells developed ADI resistance in hypoxic conditions with increased ASS1 expression. To restore ADI sensitivity as well as achieve tumor-selective delivery under hypoxia, we constructed a pH-sensitive cell penetrating peptide (CPP)-based delivery system to carry ADI inside cells to deplete both intra- and extracellular arginine. The delivery system was designed to activate the CPP-mediated internalization only at the mildly acidic pH (6.5–7) associated with the microenvironment of hypoxic tumors, thus achieving better selectivity toward tumor cells. The pH sensitivity of the CPP, HBHAc, was controlled by recombinant fusion to a histidine-glutamine (HE) oligopeptide, generating HBHAc-HE-ADI. The tumor distribution of HBHAc-HE-ADI was comparable to ADI-PEG20 in a mouse xenograft model of human breast cancer cells in vivo. In addition, HBHAc-HE-ADI showed increased in vitro cellular uptake in cells incubated in a mildly acidic pH (hypoxic conditions) compared to normal pH (normoxic conditions), which correlated with pH-sensitive in vitro cytotoxicity in hypoxic MDA-MB-231 and human prostate cancer PC3 cells. Together, we conclude that the HBHAc-HE-based peptide delivery offers a useful means to overcome hypoxia-induced resistance to ADI in breast cancer cells, and to target the mildly acidic tumor microenvironment.

Keywords: hypoxia, pH-sensitive peptide, tumor targeting, argininosuccinate synthetase, arginine deiminase resistance

Graphical Abstract

Schematic presentation of the selective intracellular delivery of recombinant arginine deiminase (ADI) using pH-sensitive cell penetrating peptides to overcome ADI resistance in hypoxic breast cancer cells MDA-MB-231.

graphic file with name nihms768214u1.jpg

Introduction

Arginine deiminase (ADI), a Mycoplasma enzyme which catalyzes the conversion of arginine into citrulline, is a promising anticancer therapeutic which shows cytotoxicity in arginine-auxotrophic tumors by depleting extracellular arginine and causing cell starvation. The pegylated form, ADI-PEG20, is currently in clinical trials for treating different cancers 1–4. Due to their low internalization, arginine depletion therapies including ADI and ADI-PEG20 work outside the cell in depleting extracellular arginine. Therefore, they are only effective in cells lacking expression of a key urea cycle enzyme, argininosuccinate synthetase 1 (ASS1), which regenerates arginine from citrulline inside the cells 5,6.

Hypoxia in solid tumors is one of the key factor driving malignant progression and therapy resistance. A hypoxic region is formed when a tumor mass outgrows the supplying range of blood vessels. The diffusion barrier causes decreased oxygen levels with increased distance from blood vessels, resulting in continuous or chronic tumor hypoxia. Besides the chronic effect, tumors can also suffer from acute hypoxia caused by collapse of immature vessels or temporary obstructions 7. In order to survive, tumor cells adapt to the hypoxic environment by regulating expression of stress-response genes 8. As a result, hypoxic tumors are more resistant to therapies and the surviving cells can act as a foci for tumor relapse, often resulting in poor prognosis 9. While the role of hypoxia in leading to resistance of tumors to radio- and chemotherapy has been well established 10,11, its effect on arginine depletion strategies has not been investigated.

In this study, we aimed to evaluate ADI-resistance under hypoxic conditions, and also to develop an ADI-delivery system to overcome resistance. Previously, we have shown that intracellular delivery of ADI using a novel cell penetrating peptide (CPP), HBHAc (KKAAPAKKAAAKKAPAKKAAAKK), can restore ADI-treatment sensitivity and enhance therapeutic efficacy in resistant ASS1-expressing cells 12,13. CPPs are a powerful tool for enabling the intracellular delivery of a wide range of cargo molecules, however their major obstacle in in vivo application is their widespread tissue distribution, leading to a low tumor:normal tissue ratio 14. Therefore, we incorporated our pH-sensitive masking peptide, histidine-glutamic acid (HE) 15–17, into HBHAc-ADI in order to improve its delivery to the tumor cells. Among different tumor targeting mechanisms, pH-sensitive design is ideal for this study since it is well established that tumor hypoxia together with the Warburg effect lead to a mildly acidic tumor microenvironment 18–21. In order to study the influence of hypoxia on ADI treatment along with pH-sensitive delivery, we designed a mildly acidic hypoxia in vitro cell model. The tumor targeted accumulation in vivo in a xenograft mouse model of human breast cancer, and pH-sensitive uptake and biological efficacy in vitro, of HBHAc-HE-ADI were evaluated.

Materials and Methods

1. Materials

HeLa, MDA-MB-231, and PC3 cells were purchased from ATCC (Manassas, Virginia, USA) and maintained in Roswell Park Memorial Institute (RPMI) 1640 media (HeLa) and Dulbecco’s Modified Eagle Medium (DMEM) (MDA-MB-231 and PC3) (Invitrogen, Carlsbad, CA, USA). Restriction enzymes were from New England BioLabs (Ipswich, MA, USA). Sephadex G25 and G50 were from GE healthcare life sciences (Piscataway, NJ, USA). HisPur™ nickel nitriloacetic acid (Ni-NTA) resin was from Thermo Fisher Scientific (Waltham, MA, USA). IRDye® 800CW (“IR800”) was from LI-COR Biosciences (Lincoln, NE, USA, USA). Radioactive 125I-Na was from Perkin Elmer (Waltham, MA). Dialysis bags with molecular weight cutoff 12–14 kDa were from Spectrum Laboratories (Rancho Dominguez, CA, USA). Matrigel™ was from BD Biosciences (Bedford, MA, USA). ADI-PEG20 was from Polaris Group (San Diego, CA, USA). pTTQ18-ADI was constructed in our laboratory as previously described 22 and pUC57 vectors were from Genomics BioSci & Tech (Taipei, Taiwan). Isopropyl β-D-1-thiogalactopyranoside (IPTG) was from Amresco (Solon, OH, USA). Phenylmethylsulfonyl fluoride (PMSF) was from EMD Millipore (Billerica, MA, USA). Fluorescein isothiocyanate (FITC), 3,3′-dihexyloxacarbocyanine iodide (DiOC6), competent E. coli BL21-T1R, protease inhibitor cocktail, Thrombin CleanCleave™ Kit, and other chemicals were from Sigma-Aldrich (St. Louis, MO, USA). Female athymic nude mice (Hsd:Athymic Nude-Foxn1nu, 4–6 weeks old) were from Harlan (Livermore, CA, USA).

2. Plasmid construction

The plasmids encoding modified ADI fusion proteins were constructed from pTTQ18-ADI. Each of the peptide sequences, poly-histidine (His6), His6-HBHAc and HBHAc-(HE)15, were added to the N-terminal of ADI. The His6 tags and HE repeats which serve as a His-tag 17 were incorporated to enable purification using Ni-NTA agarose chromatography. The His6 sequences were followed by a thrombin recognition sequence (LVPRGS) to allow for cleavage of the His-tag after production. For HBHAc-(HE)15-ADI, a short pentaglycine (G5) linker was included between HBHAc and (HE)15 to obtain flexibility, and the number of HE-repeats (n=15) was selected to obtain an HBHAc-HE sequence with an isoelectric point of 6.8 in order to target the mildly acidic pH region (pH 6.5–7). The DNA sequences of inserts were synthesized and cloned in the pUC57 vector. pTTQ18-ADI and pUC57 encoded inserts were digested with KpnI and SacI, respectively, followed by ligation. The sequences of constructed plasmids were confirmed by colony PCR and sequencing.

3. Protein expression and purification

The correct plasmids were transformed into E. coli expression strain BL21. Protein production was induced by adding IPTG. After expression, bacteria were lysed in phosphate buffered saline (PBS) containing 0.1% Triton X-100 and 2 mM PMSF, and following by centrifugation to separate supernatant from inclusion bodies. The undissolved inclusion bodies were washed and dissolved in dissolving buffer (6 M guanidine and 10 mM dithiothreitol in 50 mM Tris buffer). The dissolved protein solution was slowly dropped into 2 L of refolding buffer (1 mM ethylenediaminetetraacetic acid and 0.02% NaN3 in 10 mM phosphate buffer; for HBHAc-ADI, additional 10 mM arginine was added) with gentle stirring for 48 h at room temperature. After refolding, the aggregates were removed by passing through filter paper followed by concentration using Tangential Flow Filtration (TFF) (Millipore, Billerica, MA, USA). The concentrated fusion proteins were loaded on a Ni-NTA agarose column and then washed with 25 mM imidazole in high salt phosphate buffer (10 mM phosphate and 300 mM NaCl). The fusion proteins were then eluted with high salt phosphate buffer containing 250 mM imidazole. The excess imidazole was removed by dialysis against 10 mM phosphate buffer (pH 7.0). The His-tags were removed by Thrombin CleanCleave™ Kit per the manufacturer’s instructions. Briefly, 1 mg of His-tag containing proteins were incubated with 100 μL of thrombin-agarose resin suspension (50% v/v) in 1 mL of cleavage buffer (50 mM Tris-HCl and 10 mM CaCl2, pH 8.0) for 24 h at room temperature. Cleaved fusion proteins were recovered by collecting the supernatant after centrifuging the mixture for 5 min at 500 g. The cleaved His-tags were removed by dialysis against 10 mM phosphate buffer (pH 7.0). The purity of the fusion proteins was determined by SDS-PAGE followed by Coomassie blue staining.

4. ADI enzyme activity assay

The enzyme activity of ADI fusion proteins was determined by measuring conversion rate of arginine to citrulline using a slightly modified colorimetric citrulline quantification assay 23. Briefly, 10 μL of ADI or ADI fusion proteins were added into 45 μL of PBS and pre-warmed on a 37 °C dry bath for 5 min. Next, 5 μL of 80 mM arginine was spiked into the protein solution. After incubation at 37 °C for 15 min, the reaction was terminated by the addition of 200 μL color developing reagent (20 mM 2,3-butanedione, 0.5 mM thiosemicarbazide, 2.25 M H3PO4, 4.5 M H2SO4 and 2 mM NH4Fe(SO4)2). Finally, color was developed by heating the mixture in boiling water for 5 min followed by measuring the absorbance at 560 nm. A range of citrulline solutions (6.25 to 400 μM) was used to create a calibration curve, and the amounts of citrulline generated by ADI proteins in 15 min were determined. The enzyme activity per unit is defined as converting 1 μmol arginine to 1 μmol citrulline in 1 min at 37 °C.

5. Labeling of purified proteins

In order to track and quantify protein behavior in vitro and in vivo, purified protein samples and ADI-PEG20 were labeled with fluorescent dye or radioactive tracer. For in vitro cell assays, FITC or 125I was used; for animal imaging study, IR800 was used. The labeling reaction for fluorescent dyes was performed at room temperature for 2 h with a dye/protein molar ratio of 6:1 for FITC and 4:1 for IR800. After the reaction, unlabeled dye was removed using size exclusion chromatography (Sephadex G25) and the labeled fusion proteins were eluted with PBS. For radiolabeling, 125I was conjugated on proteins via the chloramine T method as previously described 24. The 125I-labeled proteins were purified using size exclusion chromatography (Sephadex G50), the labeled fusion proteins were eluted with PBS, and the radioactivity was determined using a gamma counter (Cora II Auto-Gamma, Packard, Downers Grove, IL, USA).

6. In vivo tumor accumulation imaging

All animal studies were performed according to the protocols approved by the University of Southern California Institutional Animal Care and Use Committee. To generate a xenograft model, 7 × 106 MDA-MB-231 cells suspended in 100 μL of Matrigel™ was subcutaneously injected into the right neck region of female athymic nude mice (Hsd:Athymic Nude-Foxn1nu, 4–6 weeks old) as previously described 15. Tumors were allowed to grow to 0.5 to 1 cm3 in volume before selection for imaging studies. The tumor-bearing mice were separated into two groups (n=3) and injected with IR800-labeled ADI-PEG20 or HBHAc-HE-ADI through tail vein. The in vivo tumor accumulation effect was imaged by IVIS SPECTRUM imaging system (PerkinElmer, Waltham, MA, USA) at 0.5, 1, 2, 4 and 6 h post-injection. After the last time point, tumor and organs were collected for ex vivo imaging.

7. Hypoxic/normoxic in vitro model

An in vitro hypoxic cell model was established in order to mimic the mildly acidic environment around hypoxic tumors. MDA-MB-231 or PC3 cells were seeded on 96-well plates with density of 5000 cells/well. After overnight incubation to allow for cell attachment, the culture medium was replaced with modified DMEM containing 50% of the NaHCO3 and L-glutamine concentration (1.85 g/L NaHCO3 and 2 mM L-glutamine) compared to the maintaining medium. The NaHCO3 and L-glutamine concentrations were reduced in order to increase the extracellular acidification rate and buffering capacity, respectively, to mimic the mildly acidic pH microenvironment of tumors 13. For the hypoxic treatment groups, the cells grown in 96-well plates were transferred to an OxyCycler C42 system (BioSpherix, Redfield, NY, USA) maintained at 0.5% O2 for MDA-MB-231 or 1% O2 for PC3 cells, and allowed to balance for 1 day. To avoid fluctuation of oxygen concentration, modified medium was pre-balanced in the respective normoxic or hypoxic incubator overnight before use. The medium pH in both normoxic and hypoxic cultures was measured every 24 h for 72 h.

8. Cellular association and uptake assay

Cell association at different pH was performed in HeLa cells using 125I-labeled fusion proteins. HeLa cells were seeded on 6-well plates at a density of 1 × 106 cells/well one day before assay. Cells were then treated with 5 μg/ml of 125I-labeled fusion proteins prepared in self-made RPMI medium (prepared from RPMI powder, without NaHCO3, with 10 mM Na2HPO4 and 10 mM citrate/citric acid) adjusted to pH 6.0, 6.5, 7.0 and 7.5. After 1 h of incubation at 37 °C, cells were washed and detached with trypsin-EDTA. The total cell association, which includes membrane association and internalization, was measured by gamma counter. Cellular uptake of ADI-PEG20 and ADI fusion proteins was also performed on hypoxic/normoxic in vitro model.

MDA-MB-231 cells were seeded in 24-well plates at a density of 5 × 104 cells/well and allowed to attach overnight, followed by incubation in either a normoxic or hypoxic incubator for 48 h. FITC labeled proteins (10 μg/mL) were then added to cell monolayers. To avoid influence on pH, sample volume was controlled in less than 1/10 fold of total medium volume. After 1 h of incubation at 37 °C, cells were washed three times with PBS and detached by trypsin-EDTA. Cellular uptake of FITC labeled protein samples was analyzed by flow cytometry (Gallios, Beckman Coulter, Brea, CA, USA)

9. Cytotoxicity assay

The acid phosphatase (ACP) assay, which avoids the effect of mitochondria dysfunction, was used to analyze cytotoxicity. MDA-MB-231 and PC3 cells were seeded on 96-well plates and prepared following the same protocol described in the hypoxic/normoxic in vitro model section. After 1 day balance in either normoxic or hypoxic incubator, ADI-PEG20 or ADI fusion proteins were spiked into culture wells at a 10-fold dilution to avoid influence on pH. Cell viability was tested 72 h post-dosing as previously described 25. Briefly, cells were washed twice with PBS, followed by the addition of 100 μL of 5 mM p-nitrophenol phosphate (p-NPP). The reaction was stopped by the addition of 10 μL of 1N NaOH. Finally, the color change was measured at 410 nm.

10. Mitochondria damage assay

3,3′-Dihexyloxacarbocyanine iodide (DiOC6), a cell-permeant, green-fluorescent lipophilic dye, accumulates in mitochondria due to their large negative membrane potential and is widely used to monitor the mitochondrial membrane potential (MMP) using flow cytometric detection 26–29. In this study, measurement of MMP was used to detect mitochondria damage. MDA-MB-231 cells were seeded on 24-well plates at a density of 5 × 104 cells/well and treated with 0.3 mU/mL ADI-PEG20 or ADI fusion proteins following the same protocol as the cytotoxicity assay. After 48 h of treatment, cells were harvested and re-suspended in 10 nM of DiOC6. The suspension was incubated at 37 °C for 15 min. Cells were then washed three times with PBS followed by flow cytometry analysis.

11. Statistical Analysis

All values in the manuscript are represented as average value ± standard deviation, and significant differences were evaluated using ANOVA, followed by the Bonferoni modified t-test. Differences with a calculated p < 0.05 were considered statistically significant, and data were marked with *, ** and *** to indicate p < 0.05, p < 0.01 and p < 0.001, respectively.

Results

1. Hypoxia associated ADI resistance in MDA-MB-231

Human breast cancer MDA-MB-231 cells are ADI sensitive with low ASS1 expression 25. To evaluate the effect of hypoxia on ADI-sensitivity, cells were treated with 0.0625 to 0.5 mU/mL of ADI-PEG20 under normoxic and hypoxic conditions, and cell viability was measured 72 h after dosing. As shown in Fig. 1A, the half maximal inhibitory concentration (IC50) of ADI-PEG20 increased from 0.25 mU/mL in normoxic condition (21% O2) to over 0.5 mU/mL in hypoxic condition (0.5% O2). In addition, it was found that ASS1 message RNA (mRNA) as well as protein abundance in MDA-MB-231 cells increased following prolonged incubation in hypoxic condition (Fig. 1B).

Fig. 1. Hypoxia-associated ADI resistance in MDA-MB-231 cells.

Fig. 1

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(A) MDA-MB-231 cells cultured in hypoxic 0.5% O2 showed resistance to ADI treatment compared to normoxic 21% O2. Cells were treated with ADI-PEG20 for 72 h. (B) Hypoxia induces ASS1 mRNA and protein expression. MDA-MB-231 cells were cultured under hypoxic conditions (0.5% O2) for the indicated time periods. Inset: MDA-MB-231 cell lysates were analyzed by SDS-PAGE followed by anti-ASS1 and anti-Actin Western blot. (A–B) Bars represent average values with error bars indicating standard deviation (n = 3). Bars marked with *, ** and *** indicate p < 0.05, p < 0.01 and p < 0.001, respectively, as determined using the Bonferoni modified t-test.

2. Design and production of ADI fusion proteins

In order to overcome hypoxia-associated ADI resistance, we designed a pH-sensitive HBHAc-HE-ADI fusion protein to achieve targeted intracellular delivery of ADI to the mildly acidic tumor region (Fig. 2A). Unmodified ADI, as well as the HBHAc-ADI fusion protein containing only the CPP sequence without the pH-sensitive masking sequence, served as non-internalizing and non-pH-sensitive controls, respectively (Fig. 2A). After successfully constructing pTTQ18 vectors encoding modified ADI fusion proteins, plasmids were transformed to E. coli BL21 strain for protein expression. Corresponding ADI fusion proteins were produced, purified, and analyzed by SDS-PAGE following with Coomassie blue staining. The protein bands showed a gradient position shift from ADI with addition of HBHAc or HBHAc-HE sequences. All three proteins had > 80% purity (Fig. 2B). Among the three proteins, ADI had highest yield of 4 mg, followed by 2.5 mg of HBHAc-HE-ADI and 0.5 mg of HBHAc-ADI, in 500 mL TB culture broth. The enzyme activity of ADI, HBHAc-ADI and HBHAc-HE-ADI was 1.2, 0.6, and 1.2 U/mg, respectively.

Fig. 2. Design, production and characterization of ADI fusion proteins.

Fig. 2

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(A) Construct design of ADI fusion proteins. Modified sequences were inserted into pTTQ18-ADI vector. (B) Production of ADI fusion proteins. The purified proteins were analyzed by Coomassie blue staining. Lane 1: Protein ladder; Lane 2: ADI; Lane 3: HBHAc-ADI; Lane 4: HBHAc-HE-ADI. (C) pH-dependent cell association of ADI fusion proteins. 125I-labeled ADI (closed squares, dashed line), HBHAc-ADI (closed squares, solid line) or HBHAc-HE-ADI (open squares, solid line) (5 μg/ml) were added to HeLa cells incubated at the indicated extracellular pH and the cell association was measured after 1 h of incubation. Data points represent average values with error bars indicating standard deviation (n = 3).

3. Cell association at different pH

Next, the pH-sensitive action of HBHAc-HE-ADI was tested on an in vitro cell model by adjusting the pH of the cell culture medium to 6.0, 6.5, 7.0 or 7.5 with lactic acid to mimic in vivo acidic tumor microenvironment. We used HeLa cells instead of MDA-MB-231 cells for this study due to their higher tolerance to abruptly changing the pH of the extracellular media. As shown in Fig. 2C, the amount of cell-associated ADI was less at all tested pHs, confirming its low binding and internalizing ability 12. With the assistance of the CPP, HBHAc-ADI showed a higher amount of cell association among all tested pHs. In addition, the association of HBHAc-HE-ADI with cells was pH-dependent. More HBHAc-HE-ADIs were associated with cell at the lower pH than the higher pH. The difference between pH 6.0 and 7.5 was 3.5-fold for HBHAc-HE-ADI and 1.6-fold for HBHAc-ADI, indicating the pH-sensitive action of HBHAc-HE-ADI (Fig. 2C).

4. In vivo tumor targeting effect

MDA-MB-231 tumor-bearing nude mice were used to test the in vivo pH-sensitive targeting effect of HBHAc-HE-ADI. We first determined if the tumor accumulation of HBHAc-HE-ADI was at least similar to the current clinical ADI formulation, ADI-PEG20. Due to its large molecular size attained by conjugation to approximately ten 20 kDa polyethylene glycol (PEG) chains, ADI-PEG20 accumulated at tumor sites, presumably through the enhanced permeability and retention (EPR) effect 30. ADI-PEG20 and HBHAc-HE-ADI were labeled with IR800 infrared fluorescent dye before injection into tumor-bearing mice through tail vein. Both HBHAc-HE-ADI and ADI-PEG20 showed a high signal at the tumor region at 30 min post-injection, and the signal intensity was visible for up to 6 h post-injection (Figs. 3A and B). These results indicated that HBHAc-HE-ADI has comparable tumor accumulation effect as ADI-PEG20. However, the ex vivo imaging of organs and tumor at 6 h after injection suggested that these two proteins were eliminated through different routes. Apart from the tumor, a high level of ADI-PEG20 was captured in the liver while HBHAc-HE-ADI was detected mainly in the kidneys (Fig. 3C).

Fig. 3. In vivo tumor targeting effect.

Fig. 3

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IR800 labeled ADI-PEG20 and HBHAc-HE-ADI were injected to MDA-MB-231 tumor-bearing nude mice through tail vein. (A) Biodistribution imaging was captured post-injection at indicated time points. White arrows indicate tumor region. (B) Signal intensity at tumor region was quantified. Bars represent average values with error bars indicating standard deviation (n = 3). (C) Ex vivo imaging of organs and tumor was performed 6 h post-injection.

5. pH changes and pH-sensitive cellular uptake in normoxic/hypoxic cultured cells

To verify the mildly acidic tumor microenvironment established by hypoxic conditions, the pH of culture media from MDA-MB-231 cells incubated under normoxic (21% O2) and hypoxic (0.5% O2) conditions was measured over a period of 72 h incubation. Under hypoxic culture condition, the pH of the medium dropped from 7.6 to 6.7 after 24 h, and continued to decrease to 6.1 after 72 h. The pH of the culture medium under normoxic condition showed a slower decrease, and was maintained at around 7.0 at 48–72 h (Fig. 4A).

Fig. 4. Effect of normoxic and hypoxic incubation conditions on pH of extracellular medium and on cellular uptake of FITC labeled protein.

Fig. 4

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(A) MDA-MB-231 cells were cultured in a normoxic (21% O2) or a hypoxic incubator (0.5% O2), and the pH of the culture medium was measured at the indicated time points. Data points represent average with error bars indicating standard deviation (n = 3). (B) FITC labeled ADI-PEG20 and ADI fusion proteins were added to the cells 48 h after incubation in normoxic/hypoxic conditions. After 1 h of incubation, cellular uptake was measured by flow cytometry and quantified by FL intensity. Bars represent average values with error bars indicating standard deviation (n = 3). Bars marked as *** indicate p < 0.001 as determined using the Bonferoni modified t-test.

After establishing the hypoxic cell culture model to create a mildly acidic environment mimicking in vivo tumor conditions, the pH-sensitive cellular uptake of ADI-PEG20 and ADI fusion proteins was determined. FITC-labeled proteins were added to cells 48 h after incubation in normoxic/hypoxic conditions (i.e. the 24 h time point indicated where the medium pH were 7.4 and 6.7, respectively in Fig. 4A). Cellular uptakes of FITC-labeled proteins were measured by flow cytometry after 1 h of incubation. As shown in Fig. 4B, cellular uptakes of ADI-PEG20 and ADI were less in both normoxic and hypoxic conditions. The uptake amount of HBHAc-ADI on the other hand was significantly higher in both incubation conditions, with only a 1.2-fold difference between normoxic versus hypoxic conditions. In contrast, the uptake of HBHAc-HE-ADI under mildly acidic hypoxic condition was 2.4-fold more than that in the neutral normoxic conditions, indicating the superior pH sensitivity of HBHAc-HE-ADI (Fig. 4B). These results were comparable to those obtained in HeLa cells (Fig. 2C).

6. pH dependent cytotoxicity in normoxic/hypoxic cells

To test the pH dependent cytotoxicity of HBHAc-HE-ADI, MDA-MB-231 cultured in normoxic or hypoxic conditions were treated with 0.0625–0.5 mU/mL of ADI-PEG20, ADI, HBHAc-ADI or HBHAc-HE-ADI. Cell viability was measured at 72 h after dosing. As shown in Fig. 5, both ADI and ADI-PEG20 exhibited similar activity, which was lower in ADI-resistant hypoxic condition (Fig. 5B) compared to normoxic condition (Fig. 5A). With the HBHAc-mediated internalization, HBHAc-ADI showed a dose-dependent, enhanced cytotoxicity under both normoxic and hypoxic conditions (Figs. 5A and B). The cytotoxicity of the pH-sensitive HBHAc-HE-ADI, on the other hand, was comparable to ADI-PEG20 and ADI under normoxic condition (Fig. 5A) where culture medium pH remained neutral (Fig. 4A). However, under the mildly acidic hypoxic condition, the cytotoxicity by HBHAc-HE-ADI increased to the level comparable to that by HBHAc-ADI (Fig. 5B). Similar results were found in PC3, human prostate cancer cells with moderate ASS1 expression 24 at doses ranging from 0.2 to 1 mU/mL (Figs. 6A and B). The mitochondrial damage induced by arginine depletion 25 at 48 h post-dosing was also determined by measuring mitochondrial membrane potential (MMP). Consistent with the cytotoxicity results, HBHAc-HE-ADI caused a higher reduction of MMP under hypoxic condition compared to normoxic condition, where there was no difference from ADI or ADI-PEG20 (Fig. 5C). Together, these results indicated that the mildly acidic pH-dependent delivery of HBHAc-HE-ADI is capable of overcoming ADI-resistance under hypoxic condition. Additionally, the cytotoxicity of ADI or ADI fusion proteins in MCF10A, pseudo-normal immortalized human breast cells, and in peripheral blood mononuclear cells (PBMCs), was minimal (Supplemental Figs. S1 and S2).

Fig. 5. Bioactivity of ADI-fusion proteins in MDA-MB-231 cells under normoxic and hypoxic conditions.

Fig. 5

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MDA-MB-231 cells cultured under hypoxic or normoxic conditions were treated with ADI-PEG20 or ADI fusion proteins. (A) Normoxic cell viability and (B) hypoxic cell viability were measured at 72 h post-dosing. (C) Mitochondria function was determined at 48 h post-dosing. Cells treated with PBS were used as a control. Bars represent average values with error bars indicating standard deviation (n = 3). (A–C) Bars marked as ** and *** indicate p < 0.01 and p < 0.001, respectively, as determined using the Bonferoni modified t-test.

Fig. 6. Bioactivity of ADI-fusion proteins in PC3 cells under normoxic and hypoxic conditions.

Fig. 6

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PC3 cells cultured under hypoxic or normoxic conditions were treated with ADI-PEG20 or ADI fusion proteins. (A) Normoxic cell viability and (B) hypoxic cell viability were measured at 72 h post-dosing. Bars represent average values with error bars indicating standard deviation (n = 3). Bars marked as * and ** indicate p < 0.05 and p < 0.01, respectively, as determined using the Bonferoni modified t-test.

Discussion

Arginine depletion therapies have become a promising therapeutic approach to treat drug resistant tumors 31. Multiple types of tumor cells have a defective arginine-to-citrulline pathway due to the suppressed expression of ASS1, thus becoming arginine auxotrophic. As such, depleting extracellular arginine by ADI and its analogs, such as ADI-PEG20, will result in inhibited cell proliferation and eventual cell death 25,32. However, a major obstacle is that these ADI-sensitive tumor cells could become resistant to arginine deprivation through ASS1 re-expression, thereby converting citrulline to arginine inside cells. The re-expression of ASS1 is regulated by external stimuli in a context-dependent manner. For example, ASS1 expression has been shown to be down-regulated by cisplatin treatment in hepatocarcinoma cell lines 33, while it is up-regulated by arginine deprivation and c-MYC in melanocarcinoma cell lines 34,35. In this report, we demonstrated that hypoxia, a common feature in solid tumors, is able to induce ASS1 expression in MD-MBA-231 cells, thereby rendering resistance to arginine deprivation-based therapy in breast cancer cells. Hypoxia-related resistance to ADI was also observed in PC3 prostate cancer cells in this study.

In normal tissues, oxygen levels are generally within a range of 4–7%, but can drop to below 2% in the hypoxic tumor region 36. In this study, we used a hypoxic incubator with 0.5–1 % of oxygen supply to mimic the tumor hypoxic environment, and evaluated its role in decreasing the effectiveness of ADI. First, we tested the effect of hypoxia on ADI treatment response in the MDA-MB-231 breast cancer cells. We found that hypoxic MDA-MB-231 cells were less sensitive to ADI treatment than their normoxic counterparts (Fig. 1A). This difference in sensitivity in normoxic versus hypoxic conditions was accompanied with an increase in ASS1 gene expression (Fig. 1B). Although the mechanism underlying hypoxia-induced upregulation of ASS1 mRNA and ADI resistance in MDA-MB-231 is still unclear, we believe it contributes to the development of resistance to ADI-based therapy. Herein, we aimed at overcoming hypoxia-induced resistance to arginine depletion in general.

The pegylated ADI analog, ADI-PEG20, has been shown to have a good safety profile, and is currently undergoing many Phase I–III clinical trials 31. However, this drug is not effective in ASS1-expressing cells due to low internalization. Previously, we have shown that a 23-mer CPP, HBHAc, can efficiently deliver ADI into cells to deplete arginine and restore sensitivity of ADI-resistant cells 12. However, the main concern with CPP-mediated delivery of bioactive cargos in vivo is poor biodistribution, leading to a lower accumulation at tumor sites and increased risk of adverse side effects at normal tissues 37. It is well accepted that both the Warburg effect and the poor vascularization as tumors enlarge will create an acidic tumor microenvironment. Using other CPPs including model amphipathic peptide and oligoarginine, we found that incorporation of an (HE)15 co-oligopeptide masking sequence renders a higher binding and uptake of the CPP in cultured cells at the mildly acidic pH 6.5–7 compared to neutral pH 7.4 15–17(R). We have also shown that the CPP-HE peptides could convey a higher tumor targeting and longer retention in vivo using an MDA-MB-231 breast cancer mouse xenograft model 15. Therefore, we incorporated the HE sequence into HBHAc-ADI to generate a novel pH-activatable HBHAc-HEADI, with improved internalization at the mildly acidic pH (Fig. 2C).

The MDA-MB-231-xenograft tumor bearing mice have previously been shown to be suitable for testing pH-sensitive targeting effect 15. The tumor targeting mechanism of HBHAc-HE-ADI depends on pH-sensitive unmasking of the HE sequence, while the current product, ADI-PEG20, is presumably based on the EPR effect as it has been reported that particles ranging from 10 to 100 nm in size or with a molecular weight lager than 60 kDa tend to accumulate inside tumors through the immature, leaky blood vessels 38. In ADI-PEG20, there are at least 10 PEGs (20 kDa each) attached, which brings the size up to around 250 kDa 39. The in vivo imaging showed that our HBHAc-HE-ADI exhibited a comparable tumor accumulation as ADI-PEG20 (Fig. 3). However, these two proteins utilized different elimination routes. HBHAc-HEADI was mainly distributed to kidney due to the smaller size of 54 kDa, which is slightly lower than renal filtration cutoff size of 60 kDa 35. Pegylation is a well-established method to enlarge molecular size to avoid renal filtration. Previous reports have shown that renal accumulation is sharply decreased with the modification of PEG size larger than 5 kDa 21. Therefore, ADI-PEG20 exhibits a relatively low signal in the kidney, but was instead eliminated mainly via the liver (Fig. 3C). This result was not surprising since the enlarged size of ADI-PEG20 is expected to cause greater uptake by the reticuloendothelial system (RES) in liver, which is responsible for macromolecule clearance 40.

In order to adapt to the hypoxic environment, tumor cells further exacerbate their Warburg metabolic phenotype by accelerating glucose uptake and glycolysis rates, which leads to increased lactic acid production and results in acidification of the extracellular environment 21. We observed a much faster acidification of the culture medium when MDA-MB-231 cells were cultured in a hypoxic environment than when maintained in a normoxic incubator. In order to study the in vitro pH-dependency of HBHAc-HE-ADI in MDA-MB-231 cells, a hypoxic exposure was utilized to generate a condition resembling the acidic tumor microenvironment. Consistent with the results in HeLa cells (Fig. 2C) and our previous studies 12, ADI-PEG20 and ADI showed low cell internalization, while HBHAc-ADI showed high internalization, in both normoxic and hypoxic culture conditions. HBHAc-HE-ADI showed a pH-dependent cell internalization, with low uptake in normoxic cells (neutral pH) and high uptake in hypoxic cells (mildly acidic) (Fig. 4B). In summary, HBHAc-HE-ADI reproducibly exhibited pH-dependency in both HeLa and MDA-MB-231 cells compared to HBHAc-ADI (Figs. 2C and 4B).

Next, the ADI-induced cytotoxicity was tested in our normoxic/hypoxic MDA-MB-231 and PC3 cell models (Figs. 5 and 6). Due to their inability to deplete intracellular arginine, ADI and ADI-PEG20 had lower cytotoxic effect in hypoxic cells, in which the ASS1 expression was induced. In contrast, the efficacy of HBHAc-ADI, with its ability of extra- and intracellular arginine depletion as well as its high internalization at both mildly acidic (hypoxic) and neutral (normoxic) pH, was not affected by O2 tension. The pH-sensitive delivery of HBHAc-HE-ADI (Figs. 2C and 4B), on the other hand, provided hypoxia-associated, pH-dependent cytotoxicity in both cell viability and mitochondria integrity assays (Figs. 5 and 6). Therefore, compared to the lack of selectivity HBHAc-ADI at different pH, HBHAc-HE-ADI shows pH-sensitive activity only in the mildly acidic pH seen in tumor microenvironments, and was inactive at neutral pH conditions at the surface of normal tissues. Thus, this pH-selective activity further minimizes the potential for undesired toxic side effects. Further, the pH-dependent targeting can also potentially improve the biodistribution of the protein drug by enhancing its delivery and/or retention at the tumor site. Interestingly, although the cellular uptake of HBHAc-ADI in normoxic and hypoxic MDA-MB-231 cells was much higher than HBHAc-HE-ADI (Fig. 4B), it did not lead to a significant enhancement in its cytotoxicity (Fig. 5). It is possible that only a small portion of ADI from the fusion protein could escape from endosomes, resulting in a comparable cytotoxicity of HBHAc-ADI and HBHAc-HE-ADI under hypoxia. Further, histidine residues in HE oligopeptide may facilitate the endosomal escape of HBHAc-HE-ADI 41,42, resulting in a larger proportion localized in the cytosol when compared to HBHAc-ADI.

Conclusion

In this report, we showed that hypoxic conditions can induce ADI-resistance through increased expression of ASS1. The intracellular delivery of ADI via HBHAc could be a useful strategy to overcome the hypoxia-induced resistance in breast and prostate cancer cells. Our pH-sensitive HBHAc-HE-ADI showed a favorable accumulation in xenografted tumors in vivo and increased in vitro tumor cellular uptake in the mildly acidic pH 6.5 compared to neutral pH 7.4. The pH-dependent internalization led to increased cytotoxicity only in hypoxic cells with a mildly acidic extracellular pH, while the activity was lower and comparable to the non-internalizing ADI and ADI-PEG20 in normoxic cells with a neutral extracellular pH. The pH-sensitive internalization and activity of HBHAC-HE-ADI provides the ability to target this biomolecule specifically to the mildly acidic pH tumor microenvironment in vivo. Therefore, HBHAc-HE-ADI fusion protein with pH-sensitive tumor targeted internalization offers a novel treatment strategy to overcome ADI-resistance induced by hypoxia, which is frequently encountered in many solid tumors, and is expected to improve treatment efficacy.

Acknowledgments

Funding Sources

This work was supported in part by grants to LJS, JLZ and DKA from the National Science Council of Taiwan (NSC 100-2320-B-002-007-MY3), the NIH National Cancer Institute (R21CA169841 and P30CA33572), and the Mary Kay Foundation (005-13). THY was a visiting graduate student at the University of Southern California (USC) from National Taiwan University. She was supported by Graduate Student Study Abroad Program (102-2917-I-002-007) from the National Science Council, Taiwan.

We would like to thank Dr. Likun Fei from the USC School of Pharmacy and Dr. Li-Peng Yap at the USC Molecular Imaging Center for assistance with establishing the mouse xenograft models and the animal imaging studies.

ABBREVIATIONS

ADI

arginine deiminase

ADI-PEG20

pegylated recombinant arginine deiminase

ASS1

argininosuccinate synthetase 1

CPP

cell penetrating peptide

HE

histidine-glutamine

HIF-1

hypoxia-inducible factor-1

Footnotes

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. THY aided in experimental design, performed experiments and wrote the manuscript. YRC aided in experimental design and performed experiments. SYC aided in performing the PC3 and PBMC experiments. WCS, DKA, JLZ and LJS aided in experimental design, analyzed the data and edited the manuscript.

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