Materials
Media and antibiotics used for the cultivation of microbes were purchased from HiMedia, Mumbai, India. Bradford reagent was purchased from Bio-Rad, Gurgaon, India. Q-Sepharose resin and Ni-NTA resin were from GE Healthcare (GE Healthcare Bio- Sciences Ltd., Uppsala, Sweden). Nitrocellulose membrane and Centricon-30 concentrator were purchased from Millipore, Billerica, MA. 10X CutSmart Buffer, RNase-A, Bgl II enzyme were procured from New England Biolabs (NEB). GeneJet gel extraction kit was purchased from Thermo Scientific. Pichia pastoris (SMD 1168) was purchased from Invitrogen, USA. AutoZyme urea detection kit is from Accurex Biomedical Pvt Ltd, Mumbai, India. E-TOXATE kit (Cat # ET0200), Amicon® Ultra-15 centrifugal filter units and 5-Fluorouracil were procured from Sigma–Aldrich, USA. Antibiotic solution (Penicillin-Streptomycin, Cat# A018), Fetal Bovine Serum (FBS, Cat #RM9955), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT, Cat #TC191), Hematoxylin solution (Mayer’s) (Cat# S058), Eosin Y solution (cat#S007) and Dulbecco's Modified Eagle's medium (DMEM, Cat #AL007A) were purchased from HiMedia (Mumbai, India). Ethanol was obtained from Labogens (Changshu, China). His-tag ELISA detection kit (Cat # L00436, Genscript USA Inc). BALB/c nude mice [CrTac: NCr-Foxn1nu (NCRNU-F)] and RMS(P) PL feed were purchased from Vivo Biotech, Hyderabad, India. Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix (Cat # A1413201) was purchased from Thermo Fisher Scientific, India. High-Capacity Endotoxin Removal Spin Columns (Cat # 88274) were purchased from Pierce Biotechnology, Rockford, USA. Recombinant human arginase I (Cat # TP304649) was purchased from OriGene Technologies, Inc. USA. Mouse anti-His monoclonal antibody was from Immunotag, USA. Surgipath Paraplast High Melt Paraffin (Cat#39601095) was purchased from Leica Biosystems, Mumbai. Materials not specified here and used in the study were of the highest analytical grade. Buffers were prepared in double-distilled water.
DNA constructs
FHA-3 encoding plasmid (pPIC9K-FHA-3) regulated under methanol induction was designed and purchased from GenScript, USA.
Expression and purification of FHA-3
The recombinant plasmid encoding FHA-3 was amplified (in E. coli DH5α cells) and was subjected to restriction digestion with Bgl II, by using a standard protocol. The digested reaction was subjected to agarose gel electrophoresis (0.8%) and the linearized pPIC9K-FHA-3 fragment was purified using GeneJet gel extraction kit. Competent Pichia pastoris cells (SMD1168) were transformed with linearized pPIC9K-FHA-3 fragment and were plated on minimal dextrose (MD) plates (1.34% (w/v) yeast nitrogen base without amino acids and without ammonium sulphate, 2% (w/v) dextrose, 1.5% (w/v) agar) and grown at 30oC for 72–96 h. Colonies obtained on the MD plates were plated on YPD (1% (w/v) yeast extract, 2% (w/v) peptone and 2% (w/v) dextrose, 1.5% (w/v) agar) plates containing an increasing concentration of Geneticin sulphate (G418) antibiotic and grown at 30oC for 4–5 days to screen for multicopy transformants. Colonies present in the plate containing the highest concentration of antibiotic were screened for the expression of recombinant protein by inoculating in 10 ml of BMGY (Buffered complex glycerol medium containing 1% (w/v) yeast extract, 2% (w/v) peptone, 1% (w/v) glycerol, 1.34% (w/v) yeast nitrogen base without amino acids and without ammonium sulphate, 0.1 M phosphate buffer pH 6.0) media (containing 50 µg/ml kanamycin and 100 µg/ml G418) and the cultures were grown for 24 h at 30oC, 250 rpm. Subsequently, 1% culture was transferred to the 500 ml of BMGY media and grown further at 30oC for 48 h. The cultures were then centrifuged to remove BMGY media and the cell mass was washed with BMMY (Buffered methanol-complex medium containing 1% (w/v) yeast extract, 2% (w/v) peptone, 1.34% (w/v) yeast nitrogen base without amino acids and without ammonium sulphate, 0.1 M phosphate buffer pH 6.0 and 1% (v/v) methanol) and resuspended in fresh BMMY media. The cultures were grown further at 30oC and induced with 1% methanol every 24 h for 5 days, which was then centrifuged to collect the media without cell mass. The presence of recombinant protein in cultivating media was determined by western blot analysis and the clones showing (maximum) expression of target protein were then selected for production of FHA-3 protein.
A single colony of the selected clone was used and the cultivation of microbe and expression of recombinant protein was done by following the procedure essentially described above. After 5 days of induction of the culture with methanol, the cultivating media was collected and subjected to affinity chromatography using (50 ml) of Ni-NTA resin. Elution of the bounded protein was done in 20 mM Tris-HCl buffer (pH 8) containing 350 mM imidazole and 250 mM NaCl and the eluted fractions were collected and analyzed for both protein content (by Bradford assay) as well as arginine-hydrolyzing activity (using AutoZyme urea detection kit form Accurex Biomedical Pvt Ltd, Mumbai, India). Qualitative analysis of these fractions was done by performing SDS-PAGE and western blot analysis. Fractions containing enzymatically active protein were pooled and dialyzed against 10 mM Tris HCl buffer containing 150 mM NaCl, pH 8.0. Dialyzed sample was then subjected to endotoxin removal step by using High-Capacity Endotoxin Removal Spin Columns and concentrated using Amicon® Ultra-15 centrifugal filter units. After adding 10% glycerol (final concentration), the concentrated protein sample was then kept at -80°C in aliquots for further use.
Enzymatic activity assay
A coupled spectrophotometric assay was used to determine the arginine-hydrolyzing activity of FHA-3 as described in Stone et al with modifications [8]. Briefly, the enzyme was activated by mixing (80 µl) of the enzyme with 20 µl of either MnSO4 or CoCl2 (final concentration 10 mM) and incubating the mixture at 50oC for 20 min. Then the activated enzyme was brought to room temperature and used in the activity assay. The arginine-hydrolyzing activity of FHA was determined by incubating activated FHA (20 µl) with 40 µl of arginine substrate (final concentration 50 mM) in 100 mM HEPES buffer of pH 8.5 or pH 7.5 (at 30oC) and the urea generated in the reaction was then determined using AutoZyme urea detection kit. The specific activity of FHA-3 (U/mg) defines the amount of FHA-3 required to convert 1 µmole of L-arginine to 1 µmole of L-ornithine and 1 µmole of urea per min at pH 8.5 or 7.5 at 30°C per mg of protein.
In vitro plasma stability of FHA-3
In vitro plasma stability of FHA-3 was determined by following the procedure described previously [25]. Briefly, blood samples were collected from healthy human volunteers in an anti-coagulant (3.8% tri-sodium citrate) containing tube, and plasma was separated by centrifugation. The protocol was approved by the Institutional Ethics Committee (IEC No: IEC/52/2021). FHA-3 protein (10 µM final concentrations) was added to the plasma and the mixtures were incubated at 25°C. Aliquots were withdrawn from the incubated mixture at designated time points and the stability of the exogenously added FHA-3 enzyme was checked by monitoring the arginine-hydrolyzing activity by using 50 mM arginine substrate. The activity of the control plasma samples (in which FHA-3 enzyme was not added) was taken as 100%.
In vivo Pharmacokinetic analysis
Male adult Sprague-Dawley (SD) rats (230–250 g; 4–6 weeks old) were procured from the Central Animal Facility of our Institute (NIPER, SAS Nagar) and were maintained under standard environmental conditions with controlled temperature (22 ± 2°C), humidity (50 ± 10%), and a 12h light/dark cycle. Animals were provided feed and water ad libitum. All animals were in good condition without any macroscopic changes in the skin and tail. Institutional Animal Ethics Committee approval was obtained in advance (IAEC No. IAEC/21/50) for the execution of research in accordance with CPCSEA rules, which are outlined in the Institute of Laboratory Animal Resources' (ILAR, USA) standards.
In vivo pharmacokinetic properties of FHA-3 were determined in SD rats by following the procedures described in the literature [16] [26], with slight modifications. Briefly, 3 mg/Kg of FHA-3 protein was administered in SD rats (n = 6) as a single intravenous dosage (in the tail vein) and ~ 0.3 ml blood samples were collected from the animals (from the retro-orbital route under anesthesia) at the designated time-points (pre-dose, 1h, 4h, 8h, 24h, 48h, 72h, 120h) in a heparinized tube. Collected blood samples were immediately centrifuged and clear plasma was separated, aliquoted, and stored at -80oC until further use. The amount of FHA-3 protein present in plasma was determined by using the His-tag ELISA detection kit (Cat# _ GenScript, USA) by following the procedure described by the manufacturer. Results obtained after ELISA were fitted into the standard curve and the amount of protein present in plasma was calculated. Non-compartment model for pharmacokinetic analysis was used to calculate pharmacokinetic parameters, Mean Residence Time (MRT) and half-life (t1/2) by using PK solver software.
Cell culture
Hepatocellular carcinoma - HepG2, prostate cancer - DU145, and lung cancer - A549 cell lines were maintained in DMEM medium with (10%) fetal bovine serum, (1%) antibiotic solution (streptomycin (100 mg/ml) and penicillin (10000 U/ml)) in an incubator with 5% CO2, 95% humidity at 37°C. Cells were allowed to grow up to approximately 80% confluency before experimentation.
Cell viability assay
The in vitro anticancer activity of FHA-3 was determined by measuring cytotoxicity against selected cancer cell lines, by following the procedure described previously [27] [28], with slight modifications. Briefly, cancer cells (3 x 105 per well) were seeded in each well of flat-bottom 24-well plates containing DMEM supplemented with 10% FBS at 37°C (in 95% humidity and 5% CO2) and allowed to grow overnight After attaining 80% confluence, FHA-3 was added in designated concentration (0–50 U/ml final concentration) to the cells and the plates were further incubated for 72 h at 37oC and viability of the cells was determined by MTT assay [29]. For this, MTT solution (final concentration of 0.5 mg/mL) was added to the well and the plates were further incubated for 4 h at 37°C to induce reaction. After removing the supernatant, formazan crystal was dissolved in Dimethyl sulfoxide (DMSO − 500 µL) and the absorbance was measured at a dual-wavelength of 550 nm and 630 nm using a multi-mode automated microplate reader (Flex station III, Molecular Devices, Sunnyvale, CA, USA). The results were expressed as percentage cell viability, assuming the viability of untreated control cells as 100%. The concentration of enzymes required for 50% inhibition of the cells in culture was defined as the IC50, and IC50 values were determined by using GraphPad Prism 6 (GraphPad Software, Inc.). Two independent experiments were performed for each study and all measurements were performed in triplicates. Data were represented as mean ± S.D.
Xenograft nude mice tumor model
BALB/c nude mice [CrTac: NCr-Foxn1nu (NCRNU-F)] and RMS(P) PL feed were purchased from the Vivo Bio Tech Ltd., Hyderabad, India, and transferred to a pathogen-free environment of National Toxicology Center (NTC) in NIPER, SAS Nagar. The animals were housed in sterile and clean cages with HEPA filters with free access to diet and water under controlled conditions of temperature: 25°C, humidity: 50%; and a 12 h light/dark cycle. All the animals were acclimatized for one week before the start of experiments and were properly maintained by taking all necessary precautions and care.
In vivo anticancer activity of FHA-3 was determined in the hepatocellular carcinoma (HCC) xenograft model by following the procedures as described previously [27] [28], with slight modifications. Briefly, 2x106 viable HepG2 cells were dispersed in 1:1 (v/v) Phosphate buffered saline (PBS) and matrigel in a final volume of 100 µl and injected subcutaneously into the right flanks of each mouse (3–4 weeks old BALB/c nude mice) and the tumors were allowed to grow in the animals. The sizes of tumors were measured individually with a Vernier Caliper twice a week. After attaining the desired tumor volume (> 80 mm3), tumor-bearing animals were randomly divided into 4 groups (n = 7) and treated with indicated samples (0.9% saline solution, 5-FU (10 mg/kg/mouse once a week), FHA-3 (300 U/mouse twice a week) and a combination of FHA-3 + 5-FU) by injecting the sample intraperitoneally for three weeks. Tumor dimensions were measured in situ during the treatment by Caliper measurement and tumor volume (0.5 x L x W2) was calculated and plotted. After the completion of the treatment period, the animals were sacrificed (by CO2 inhalation) and the tumors were excised and weighed.
Histological examination of tumor tissue
Histological examination of extracted tumor tissue was done by following the procedure described previously [27][28]. Briefly, after the completion of the treatment schedule, animals were sacrificed and tumors were isolated from each animal, sliced, and fixed in 10% v/v formal saline. Thereafter, the tumors were subsequently embedded in paraffin; sections of 5 µm were prepared and mounted on pre-coated slides. Sections were stained with hematoxylin and eosin to observe the structural changes. A cover slip was mounted on the stained sections using DPX and observed at 40x magnification using the OLYMPUS BX51 microscope. The microscopic images were captured with the OLYMPUS DP 72 camera attached to the microscope.
Statistical analysis
The data obtained in the results are expressed as mean ± S.D. The statistical analysis was performed using GraphPad Prism 8. The t-test was done for statistical comparison between two groups, whereas the one-way analysis of variance (ANOVA) followed by Tukey’s test was performed for the comparison between more than two different groups. A p-value of less than 0.05 was considered to be significant.