2.3. Measurement of Anticancer Activity In Vivo

Thirty-six male BALB/c mice, 6–8 weeks old, with an average body weight of 20–25 g, were supplied by the Animal House, Faculty of Veterinary Medicine, Universiti Putra Malaysia (UPM). The animals were kept in 12 h light and dark cycles at a controlled temperature of 25 ± 2°C and fed regularly with mouse chow. Mice were housed for a week under these conditions to allow acclimatization before treatment. The study was approved by the Animal Care and Use Committee (ACUC), Faculty of Veterinary Medicine, Universiti Putra Malaysia (ref.: UPM/IACUC/AUP-R055/2017).

Mice were allotted into 6 groups (n = 6), namely, I: negative control, II: cancer control, III: treatment 1, IV: treatment 2, V: blank Pd-NP, and VI: positive control. Leukemia allografts were then established by injecting 1 × 10⁶ WEHI-3B cells suspended in 300 μl ice-cold PBS intraperitoneally into the abdomen of each mouse except those in group I. Mice were tested daily for the development of leukemia by the detection of leukemic cells in a tail vein blood smear using Wright stain. Once leukemia was established, several days after the injection of WEHI-3B cells, treatments were administered daily for 4 weeks by oral dosage of 300 μl after 12 h without food using a syringe and ball-tip stainless-steel needle. Group II received saline only; group III received Pd@W.tea-NPs at 50 mg/kg; group IV received Pd@W.tea-NPs at 100 mg/kg; group V received Blank Pd-NPs at 100 mg/kg; and group VI received ATRA (5 mg/kg). Group I received no oral dose. The condition of animals was monitored daily. At the end of the study, animals were anesthetized by intraperitoneal injection of ketamine (83 mg/g) and xylazine (13 mg/g) and then sacrificed. The spleen was collected for macroscopic and microscopic analysis.

Blood samples were collected by heart puncture. Serum was collected and analyzed immediately using standard diagnostic kits (Roche) in an automatic biochemistry analyser (Hitachi 902, Japan).

The size of the spleen collected from each animal was measured immediately using a caliper, and then, the spleens were weighed and photographed.

Spleens were washed with normal saline, fixed in 10% formalin, passed through ascending concentrations of ethanol (50, 70, 90, and 100%), embedded in paraffin wax, and then sectioned. Before staining, sections were cleared of wax in 2 changes of xylene for 3 min each, hydrated by passing through descending concentrations of ethanol (2 changes each at 100, then 70%), and rinsed with water for 3 min. Sections were then stained with hematoxylin and eosin (H&E). After staining, sections were dehydrated in ascending concentrations of ethanol (2 changes each for 3 min at 70, then 100%) and then cleared in 2 changes of xylene for 3 min each. Histopathological features, comprising vessel congestion, red blood cell extravasation, hematoma, cell necrosis, nuclear changes, and fibrosis, were scored in 4 sections from each mouse by a blinded observer, and the spleens were graded as 0 (no distinguishable change); 1 (mild change—up to 30%); 2 (moderate change—31–60%); and 3 (severe change—61–100%). Data were analyzed using the Mann–Whitney test (SPSS 16.0), taking P < 0.05 as significant change.

The Dako Envision® + Dual Link System-HRP (DAB+) kit (Dako K4965, USA) was used, with a slight modification of the protocol, to detect apoptotic cells in the spleen. Tissue sections, prepared as above, were deparaffinized, rehydrated with ascending concentrations of ethanol (100, 90, and 70%), and washed in distilled water. Wax-enclosed sections were then flooded with dual endogenous enzyme (Dako K4065, USA) as a blocking agent and incubated for 10 min. Sections were then washed with citrate buffer solution (10 mM, pH 6.0) (Sigma, USA), immersed in tris-buffered saline with Tween-20 (TBST) for 3 min, and then incubated with CD3 primary antibody (T-lymphocyte marker) (Abcam ab5690, UK) or CD19 primary antibody (B-lymphocyte marker) (Bioss bs0079R, USA) at 4°C overnight. After washing with TBST, sections were incubated for 45 min with labeled polymer-HRP reagent (Dako K4065, USA) and then washed again with TBST. DAB + substrate-chromogen solution (Dako K4065, USA) was then applied for 3 min. Finally, sections were counterstained with Myer's hematoxylin, mounted in DPX medium, and observed under a light microscope at 40x magnification.

Total protein was extracted from splenic tissues using RIPA buffer (Sigma-Aldrich, USA) supplemented with protease inhibitor cocktail (Sigma Aldrich Co, LLC, USA). Samples of 20 μg were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to PVDF membranes. Membranes were probed using specific primary antibodies against GAPDH (Abcam ab8245), Bcl-2 (Abcam ab59348), Bax (Abcam ab32503), or cytochrome-C (Abcam ab13575) at 4°C overnight. After washing 3 times (for 5 min each) in TBST, the ALP-conjugated goat anti-rabbit secondary antibody (Bio-Rad ab20571 for anti-Bcl-2 and anti-Bax primaries) or goat anti-mouse secondary antibody (Bio-Rad 170-6464 for anti-GAPDH anti-cytochrome-C primary) was applied, and membranes were then incubated with NBT-BCIP buffer for 15 min in the dark. Protein bands were visualized and captured using a Bio-Rad Gel Doc system and quantified by densitometry using molecular imaging software (ImageJ 148-JDK 6 software).

Spleens were placed in RNAlater (Ambion, USA) overnight at 4°C before being minced and then stored in a freezer at −80°C. Tissues were snap-frozen in liquid nitrogen and crushed to a powder before total RNA was extracted using the Qiagen RNAeasy Mini Kit (Qiagen, Germany) and quantified using a nanospectrophotometer (Beckton Coulter, USA). Adequate purity was confirmed based on A260/A280 ratios of 1.8–2.1. Reverse transcription to cDNA was achieved using the Maxima First Strand cDNA synthesis kit (Thermo Scientific, USA). Quantity of Bax, Bcl-2, cytochrome-C, and GAPDH mRNA was measured using SYBR Select Master Mix (Life Technologies, USA) in an Eco Illumina instrument (Illumina, USA) using thermal cycling parameters (optimised for each primer pair) of 95°C for 10 min and then 40 cycles of 95°C for 15 s and 55–60°C for 15–30 s. Quantity of Bax, Bcl-2, or cytochrome-C mRNA was expressed in relation to GAPDH using standard curves for each mRNA and a delta-Ct relative quantification model with PCR efficiency correction using Eco Study software (Illumina, USA). The target cDNA was amplified using the following primers: Bcl-2-forward 5′-CCAGACTCATTCAACCAGACA-3′ and reverse 5′-GATGACTGAGTACCTGAACCG-3′; Bax-forward 5′-TTTGCTACAGGGTTTCAT-3′ and reverse 5′-CTCCATATTGCTGTCCAG-3′; cyt-C forward 5′-GTCTTATGCTTGCCTCCCTT-3′ and reverse 5′-CGTCTGTCTTCGAGTCCGA-3′; and GAPDH forward 5′-CGGGACCTAATGAAACTCCA -3′ and reverse 5′-AATCTCCACTTTGCCACTGC-3′.