Isolation and screening of Cytosine deaminase-producing fungi
For isolation of CDA-producing fungi, different soil samples were collected from Monufia, Qaluobia, and Sharqia Governorates, Egypt, and used fungal isolation on Czapekʼs-Dox Agar medium, incubated for 7 days at 45 °C [42]. For screening for CDA-producing potency, a plug from each homogenous axenic fungal culture was inoculated into 50 ml Czapekʼs-Dox broth medium/ 250 ml Erlenmeyer conical flasks, incubated at 45 °C for 7 days. The fungal mycelia were collected by filtration, the pellets were washed with sterile distilled water, and their intracellular crude proteins were extracted [29, 30]. Five grams of each fresh fungal biomass were grinded into fine powder in liquid nitrogen, then dispensed to 5 ml Tris-HCl buffer (pH 7.0, 50 mM) with 1 mM phenylmethansulphonyl floride (PMSF), 10 μl β- mercaptoethanol and 1 mM CaCl2 [42,43,44]. The mixture was shaken for 10 min by vortex and centrifuged at 8000 rpm for 10 min at 4 °C. The supernatant was used as a crude source of CDA. The activity and protein concentration were determined.
Cytosine deaminase (CDA) activity
The deamination activity of CDA was assayed as described by Sakai et al. [44, 45] with slight modifications. The reaction mixture contains 100 mM cytosine and 0.5 ml of enzyme in Tris-HCl buffer (pH 7.0, 50 mM) in 1 ml total volume. Blanks of enzyme and substrate were prepared separately. The reaction was incubated for 20 min at 37 °C and stopped by 10% TCA. The concentration of released uracil was assessed at 286 nm using blanks of cytosine and enzyme separately as baselines. Authentic concentrations of uracil were measured at the same conditions, and used for the calculation of enzymatic activity. One unit of CDA was expressed by the amount of enzyme releasing 1 mM of uracil from cytosine per min under standard assay conditions.
The protein concentration was measured by Folin’s reagent [46], compared to the authentic concentration of bovine serum albumin.
Morphological and molecular identification of the potent fungal isolates
The potent CDA-producing fungal isolate was identified based on their morphological features according to the universal keys [22, 23, 47,48,49,50]. The identification of the potent fungi was confirmed from the sequence of internal transcribed spacers (ITS) region [24, 51, 52]. Briefly, 0.2 g of the mycelia was pulverized in liquid nitrogen, vigorously homogenized, 500 μl of extraction buffer (2% CTAB, 2% PVP40, 0.2% 2-mercaptoethanol, 20 mM EDTA, 1.4 M NaCl in 100 mM Tris-HCl, pH 8.0) was added. Genomic DNA (gDNA) was used as a PCR template with primers ITS4 5′-GGAAGTAAAAGTCGTAACAAGG-3′ and ITS5 5′-TCCTCCGCTTATTGATATGC-3′. The PCR reaction contains 10 μl of 2x master mixture (i- Taq™, iNtRON Biotech.), 2 μl gDNA, and 1 μl of each primer in 20 μl total volume. The amplicons were analyzed with 1% agarose gel in 1× TBE buffer using a DNA marker, purified, and sequenced. The obtained ITS sequences were searched using the BLAST tool with non-redundant sequences on the NCBI database. MEGA X software package was used for multiple sequence alignments, the FASTA sequences were imported, and aligned by the Clustal W algorithm [53], then the phylogenetic relatedness of the target sequences was constructed with 250 bootstrap replication [54].
Purification, molecular subunit structure, and peptide fingerprint of CDA from a. niger
The potent fungus was grown on Czapek’s-Dox, mycelia were collected, and washed in sterile Tris-HCl buffer (50 mM, pH 7.0). Fungal pellets (50 g) were grinded into fine powder in liquid nitrogen and suspended in 50 ml Tris-HCl buffer of 1 mM EDTA, 1 mM PMSF, 1 mM β-mercaptoethanol and 1 mM CaCl2. After homogenization, the mix was shacked vigorously by vortex for 10 min then centrifuged for 10 min at 10,000 rpm to remove the cell debris. Dialyzer membrane of 20 kDa cut-off (Cat#546–00051) was used to concentrate the crude protein against PEG6000 [55]. The concentrated CDA preparations were further purified by gel-filtration and ion-exchange chromatographic techniques [56, 57] The most active fractions were collected, based on their activity for the subsequent biochemical characterization, and molecular mass homogeneity by SDS-PAGE analysis. The homogeneity and molecular subunit structure of the purified CDA were assessed by SDS-PAGE [58], normalizing to authentic protein marker. While, the molecular mass of the entire purified CDA was assessed by native-PAGE, without SDS on the sample and running buffers [23, 30].
The peptide fingerprint of the purified CDA was analyzed by the Liquid Chromatography-Tandem Mass Spectrometry of nanospray ionization (LC-MS/MS) at the Proteomics and Metabolomics Facility Core, 57,357 Children’s Cancer Hospital Foundation, Egypt. The SDS-PAGE gel band containing the putative CDA was excised, grinded, and destained by 200 μl of 50 mM ammonium bicarbonate (AB)-acetonitrile (1:1 v/v) [23]. The excess acetonitrile was removed by vacuum, the gel was re-swelled in 100 mM AB with 10 mM DTT for 30 min, followed by 100 mM AB with 50 mM iodoacetamide. The dried gel pieces were digested with trypsin for 12 h at 37 °C [30], the supernatant was pooled, dissolved in 100 μl of extraction buffer (5% formic acid/ acetonitrile, 1:2 v/v), and incubated for 15 min at 37 °C. The peptides were desalted and analyzed by nanospray ionization with Triple-TOF 5600 hybrid mass spectrometer, interfaced by nano-scale RP-HPLC [33, 59]. A linear gradient of acetonitrile (ACN) buffer (5–60%) was used for peptide elution from the column to mass spectrometry, with independent acquiring MS/MS data from m/z for 50–2000 Da. The raw MS/MS data were extracted, and the peptides were identified by Protein Pilot 4.0 (ABSCIEX) normalizing to the proteome of Aspergillus niger.
Conjugation of the thermostable a. niger CDA with dextran
Sodium periodate-activated dextran was cross-linked with the purified CDA for 24 h at 4 °C, in presence of trimethylaminoborane (0.15 M) [60]. The Schiff base developed from CDA reactive amino groups and dextran aldehyde groups were stabilized by reduction with 3 mg/ml sodium borohydride [43]. Different molar ratios of purified CDA (0.067, 0.135, and 0.203 mM) and activated dextran (40 mM) were examined. The activity of free CDA and Dextran-CDA conjugates were assessed by the standard assay. The immobilization yield (%) of CDA was expressed by the ratio of the activity of conjugated CDA to the free- CDA × 100.
Modification of the surface reactive groups of a. niger CDA upon dextran conjugation
The total surface reactive amino groups of the free and Dextran-CDA conjugates were assessed by Ninhydrin reagent [61]. The free CDA and Dextran-CDA conjugates (1.0 mg/ ml) were amended with 100 μl of Ninhydrin reagent, boiled for 5 min, and the developed blue complex was measured at A575 nm, compared to controls of activated dextran. The total surface reactive amino groups were expressed by the intensity of developed color at A575 nm of Dextran-CDA conjugates to the free enzyme × 100. The total surface reactive thiols of free and Dextran-CDA conjugates were determined by Ellman’s reagent (DTNB) [62] with slight modifications [24, 30, 43]. The enzyme (free and conjugated) were amended with 100 μl of 10% SDS, incubated for 10 min, and then 50 μl of 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) (25 mM) was added, vortex and incubated for 30 min. The intensity of the developed yellow complex was measured at A420 nm, and the modification ratio of the surface thiols of CDA was expressed by the color absorbance of CDA-dextran conjugates to the absorbance of free CDA × 100.
Proteolytic mapping of free and dextran-CDA conjugates
The structural stability of the free and CDA-dextran conjugates in vitro in response to proteolysis by proteinase K and trypsin were assessed [22, 23]. The native and CDA-dextran conjugates (0.8 mg/ml) were incubated with Trypsin and proteinase K (10 μmol/ min/mg) at 37 °C for 1 h, then PMSF (1 mM) was added to stop the proteolytic activity, and the remaining CDA activities were assessed by the standard assays.
Biochemical properties of the free and dextran-CDA conjugates
The optimal reaction temperature of the free and CDA-Dextran conjugates was assessed by incubation of the reaction at 30, 37, 45, 50, and 55 °C, and the enzyme activity was determined by the standard method. To determine the enzyme thermal stability, enzymes were pre-incubated without substrate at different temperatures as 30, 37, 45, 50, and 55 °C, and the residual activities of the enzymes were assessed after 30, 60, 90, and 120 min by standard assay [22,23,24,25]. The enzyme thermal kinetic parameters such as half-life time (T1/2), thermal inactivation rate (Kr), and stabilization folds were assessed [22]. The effect of reaction pH (3.0–10.0) on the activity of free and Dextran-CDA conjugates was assessed in citrate-phosphate buffer (50 mM, pH 3.0–5.0), and Tris-HCl buffer (50 mM, pH 6.0–10.0), the reactions were incubated, and enzyme activities were determined. The pH stability was assessed by pre-incubating the enzyme at different pH (3.0–10.0) at 4 °C for 1.5 and 3.0 h, then measuring their residual activities by the standard assay [24, 25]. The pH precipitation profile of free CDA and Dextran-CDA conjugates were assessed by incubating the enzymes at a pH range (3.0 to 10.0) at 4 °C for 24 h, then centrifugation for 10 min at 10,000 rpm, and the precipitated proteins were collected and measured by Folin’s reagent [46]. The isoelectric point (pI) was defined by the pH at which maximal protein precipitation was obtained [56, 57]. The effect of inhibitors on the activity of free and Dextran-CDA conjugates was evaluated. The enzymes were desalted by dialysis against Tris-HCl buffer (pH 7.0, 50 mM) with 1 mM EDTA for 2 h. Different cations such as K2+, Ba2+, Fe3+, Hg2+, Ca2+, Al3+, k+, Na+, and Cu2+ were added to the enzymes at 1 mM concentration for 2 h at 4 °C, the substrate was added, and the activity of enzymes was measured by the standard assay. The effect of different suicide amino acid reactive analogues “hydroxylamine, iodoacetamide, guanidine thiocyanate, 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB), 3-methyl-2-benzothiazolinone hydrazone (MBTH) and hydrogen peroxide (H2O2)” on the activity of free CDA and Dextran-CDA conjugates were measured, the mixtures were incubated at 4 °C for 2 h, then the residual activity of enzymes was measured.
Substrate specificity and kinetic parameters of the free and CDA-dextran conjugates
The affinity of free and modified enzymes towards various substrates; L-asparagine, L-tyrosine, L-arginine, L-methionine, L-cysteine, L-phenylalanine, L-glycine, and L-tryptophan has been evaluated comparing to cytosine as standard substrate at a final concentration (10 mM). The affinity of free and modified CDA for deamination of 5-flurocytosine as substrate was assessed [63, 64], and the concentration of released 5-flurouracil was calculated from their authentic concentrations at the same conditions. The enzyme kinetic properties such as Michalis-Menten constant (Km), maximum velocity (Vmax), turnover number (Kcat), and catalytic efficiency (Kcat/Km) of the free and CDA-dextran conjugates were determined by the GraphPad Prism Software Package [25, 30].
In vitro anticancer activity
The antiproliferative activity of the native and CDA-Dextran conjugates was evaluated towards different tumor cell lines; liver carcinoma (HepG-2), breast carcinoma (MDA-MB-231), and prostate cancer (PC-3) regarding the normal cells Oral Epithelial cells (OEC) with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay [65]. The 96-well plate was seeded with 103 cells/well and incubated overnight at 37 °C in a CO2 incubator, the prodrug 5-fluorocytosine was amended with different concentrations (0, 10, 20, 30 μg/ml)/ well, and the plate was incubated for 5 h, then different concentrations of each enzyme was added, then incubation was continued for 48 h at the same condition. The MTT reagent was added, and the developed purple formazan complex was measured at λ570 nm. The IC50 value was expressed by CDA activity reducing the initial number of tumor cells by 50%, regarding phosphate buffered saline.
In vivo pharmacokinetics of native and CDA-dextran conjugates
The pharmacokinetic properties of the free and dextran conjugated enzymes were determined using male mice (25 g of 30 days old). The in vivo experiments were performed according to the guidelines of the Institutional Animal Care and Use Committee of the Faculty of Medicine, Zagazig University, and NIH guidelines under protocol 15–08-263. Prior to injection, the mice were acclimatized for 5 days. The experimental mice were grouped into five groups: 1- Negative control, mice free from Ehrlich Ascites Carcinoma (Ehrlich cells, EAC), 2- positive control, mice subcutaneously injected with 2.5 × 106 of EAC cells, incubated for 5 days till the tumor size reached about 50 mm3, inoculated with 1 μM of 5-fluorocytosine (5-FC), 3- Native-CDA group, the eight days post-inoculated mice of EAC with 5-FC, were injected with single dose of the free CDA, 4-CDA-Dextran conjugates group, the eight days EAC post-inoculated mice with 5-FC were injected with a single dose of CDA-dextran conjugates. 5- Native-CDA treated samples, the eight days EAC post-inoculated mice without 5-FC, were injected with a single dose of native CDA without 5-FC. 6-CDA-Dextran treated samples, the eight days EAC post-inoculated mice without 5-FC, were injected with a single dose of CDA-dextran. At the end of the experiment, the mice were anesthetized with urethane (1 g/kg body weight) and sacrificed by cervical dislocation [66–69]. The remaining animals (5 in each group) were kept for evaluating the survival percentage (life span prolongation). Blood samples were withdrawn by cardiac puncture from all animal groups in tubes containing EDTA for hematological assays. Hemoglobin (HB), counts of white blood cells (WBCs), and red blood cells (RBCs) were analyzed by the standard automated assay.
The Ehrlich solid tumors were harvested from each mouse and rinsed with saline and various biochemical parameters, and histopathological and immunohistochemical analyses were conducted. For the biochemical parameters, the activity of poly [ADP-ribose] polymerase 1(PARP-1) was assessed using Mouse Poly [ADP-ribose] Polymerase 1 (PARP) ELISA Kit (Cat # MBS918279). The titer of apoptosis-inducing factor (AIF) was determined by Mouse AIF ELISA kit (Cat. # EM0826). The concentration of malondialdehyde (MDA), nitric oxide (NO), and reduced glutathione (GSH) were determined by the Biodiagnostic Kit.
The tumor volume and weight were analyzed: the tumor volume was measured by the caliper.
The volume was expressed in mm3 using the formula V = 0.5a x b2, where a and b are the short and long diameters, respectively.
The life span prolongation was determined according to the formula:
Increase in the life span ILS% = (T-C)/C × 100, Where T is the median survival time of treated mice, and C is the median survival time of positive control mice.
Part of the solid Ehrlich tumor along with liver tissue were fixed in 10% formalin embedded in paraffin and stained by hematoxylin and eosin (H and E) stain. Sections were microscopically examined and photographed. As well as, immunohistochemical staining analysis of the solid Ehrlich tumor was performed for investigating the cyclin D1 activity. Tissue sections (3–5 μm) were deparaffinized in xylene, slides were incubated for 10 min in 3% H2O2 to block endogenous peroxidase. Antibody binding was detected by Dako’s HRP Envision kit (DakoCytomation, Denmark). Tissue samples were incubated with primary antibody (Anti-Cyclin D1 antibody, ab16663, Abcam, UK, diluted in 1/100 PBS) for 1 h. The intensity of the staining areas was expressed as follows; grade-0, a total absence of staining or < 5% of cells stained; grade-1, mild to moderate nuclear staining (5–50% cells stained); grade-2, strong nuclear staining (> 50% cells stained).
Deposition of the fungal isolate
The sequence of the most potent CDA CDA-producing fungal isolates Aspergillus niger was deposited into Genbank with accession # MW332264.1.