Protease Inhibitor Cocktail Set III was obtained from Calbiochem (Darmstadt, Germany); CD-31 (PECAM-1) was obtained from Invitrogen (Carlsbad, CA, USA) and Dulbeccos modified Eagles medium (DMEM) and fetal calf serum (FCS) were obtained from Gibco-Invitrogen (Paisley, UK). Bovine serum albumin (BSA), streptavidin-horseradish peroxidase, 3,3,5,5-tetramethylbenzidine (TMB) and poly-L-lysine solution were obtained from Sigma-Aldrich (St. Louis, MO, USA). Taq DNA polymerase was obtained from Invitrogen. Oligonucleotide primers and Tris buffer were obtained from Sigma-Aldrich and dNTPs from Fermentas (Waltham, MA, USA). 3-3-Diaminobenzidine chromogen/H₂O₂ substrate in buffer solution (pH 7.5) (DAB kit) and rabbit anti-SRL polyclonal antisera were obtained from Bangalore Genei India Pvt. Ltd. (Bangalore India). Caspase-3 (3G2) mouse mAb was procured from Cell Signaling Technology, Inc. (Danvers, MA, USA). Species-specific HRP-labelled secondary antibodies were procured from Bio-Rad Laboratories (Hercules, CA, USA). Anticoagulated tubes were obtained from Greiener Bio-One (1 ml K3E K3EDTA; Frickenhausen, Germany) and a flat bottom 96-well micro-test plate were obtained from Tarsons (Kolkata, India). All other reagents were of analytical grade and Milli-Q water was used for all the preparations.

Purification of SRL from the sclerotial bodies was carried out as previously described (12). Briefly, SRL was extracted from powdered sclerotial bodies with 50 mM acetate buffer containing 100 mM NaCl (pH 4.3) and subjected to 30% methanol precipitation followed by ion exchange chromatography on CM cellulose matrix and finally purified on a Superdex G-75 gel filtration column. The purified lectin was used for biodistribution and efficacy studies. The lectin activity was routinely determined by the 2-fold serial dilution method using trypsinised human erythrocytes.

Custom designed anti-SRL polyclonal antibodies were purified from immunized rabbit serum (from Bangalore Genei India) by subjecting to 50% ammonium sulphate precipitation, followed by affinity chromatography on a Protein A agarose column equilibrated with 100 mM phosphate buffer (pH 7.2) containing 150 mM NaCl phosphate-buffered saline (PBS). Bound SRL antibodies were eluted with 100 mM glycine-HCl (pH 2.5), dialysed against PBS and stored at −20°C.

Human colon cancer HT29 cells were obtained from the European Cell Culture Collection via the Public Health Laboratory Service (Porton Down, UK). HT29 cells were cultured in DMEM supplemented with 10% FBS, 100 units/ml penicillin and 100 µg/ml streptomycin (complete DMEM) at 37°C in 5% CO₂.

All animal experiments were carried out at the Advanced Centre for Treatment, Research and Education in Cancer with approval from the Ethics Committee (IRB approval no. 07/2012) of Tata Memorial Centre-ACTREC-IAEC (Institutional Animal Ethics Committee). Inbred female BALB/c nude mice (6–8 weeks old, weighing 18–22 g) for efficacy studies and male Swiss albino mice (6–8 weeks old, weighing 20–25 g) for pharmacokinetics and biodistribution studies were used. The animals were housed in groups of 4–5 per polycarbonate cage under individually ventilated caging systems (IVCs) under controlled conditions of 55±5% humidity, 22±2°C temperature, and light cycle (12:12 h). All mice were provided with easy access to in-house pelleted feed and with ad libitum UV-treated water and were monitored daily for general health status.

HT29 cells were suspended at 1×10⁷ cells/ml in DMEM and a cell suspension (0.1 ml) was injected subcutaneously into two donor mice. After 4 weeks, the tumours from these donor mice were excised, chopped into 2- to 3-mm fragments and a single piece of tumour was transplanted subcutaneously into each of 12 BALB/c nude mice. Tumours were allowed to grow to reach the appropriate size of 5–6 mm, i.e. 80–120 mm³ in volume and animals were divided randomly into 2 groups of 5 mice in each group, one for SRL treatment (T) and other used as a control (TBS, C). Mice were intraperitoneally administered SRL (30 mg/kg body weight/mouse) in TBS (pH 7.5) on every other days for 17 days for a total of 9 doses. The mice in the control group were injected intraperitoneally with TBS at the same units (5–10 UI) and maintained for 2 weeks after the last dose. The mice were weighed and tumour volume was also determined by measuring the length (L) and width (W) of the tumour after each injection. The tumour volume at day n (TVn) was calculated as TV (mm³) = (L×W²)/2. The relative tumour volume at day n (RTVn) vs. day 0 was expressed according to the formula: RTVn = TVn/TV0 (21,22). Regression of tumour T/C (%) in the treated vs. control mice was calculated using: T/C (%) = (mean RTV of treated/control group) × 100. The changes in the tumour size of the treated mice at different time-points compared to the control were used for monitoring the efficacy of the lectin. Mice were sacrificed 2 weeks after the last dose, and whole blood was collected in tubes coated with anticoagulant and used for analysis of total blood cell count, hemoglobin content, WBC and platelet count. Tumours were excised and organs such as liver, kidney and lung were collected directly in PBS to remove the blood traces, wrapped in aluminium foil and stored at −80°C until further use for immunohistological analysis. Excised tumours were also snap frozen and stored at −80°C for STR analysis.

In order to ascertain the human origin of the tumours using short tandem repeat (STR) method (23), a small piece of the tumour tissues was collected from BALB/c mice with HT-29 xenograft, excised after 2 weeks from the last dose of injection. These tissues were snap frozen and stored at −80°C for extraction of DNA. DNA was extracted from the samples using phenol chloroform extraction followed by ethanol precipitation. The DNA precipitates were suspended in Tris-EDTA (TE) buffer, pH 8.4. The extracted DNA was subjected to PCR amplification using human-specific microsatellite primers (forward, 5′-TGCCATTTGTGGGTACATTC-3′ and reverse, 5′-TTGTGTTTCTTTTTCTGTTCCTACA-3′). In brief, 100 ng of target DNA was amplified in a 15-µl reaction volume containing 5 IU of Taq DNA polymerase, 0.4 pM each oligonucleotide primer, 10 mM Tris buffer, 3.5 mM MgCl₂, 0.4 mM each of dNTPs. PCR was performed with a 5-min denaturation at 94°C followed by 34 cycles of a 20-sec denaturation at 94°C, a 20-sec annealing at 58°C, and a 30-sec elongation at 72°C in a thermal cycler (Eppendorf, Hauppauge, NY, USA). Amplicon was separated on 2% agarose gel and visualized by UV light.

Swiss albino mice were randomized into two groups, the control group (TBS) and the SRL-treated group which included 4 time-points (1, 6, 24 and 48 h) with 3 animals for each time-point. Lectin was prepared in TBS (pH 7.5), hence TBS served as the vehicle control. Animals were injected intraperitoneally with TBS and SRL (24 mg/kg body weight/mouse) and maintained for different time-points and blood was collected from the treated and control mice by retro-orbital bleeding. After incubation of the blood at room temperature (RT) for 15 min, serum was collected by centrifuging the sample at 2,500 rpm for 15 min at 4°C and stored at −80°C until further use. The pharmacokinetic profile of the serum samples was evaluated by ELISA. Serum samples collected at 24 h were also analysed for biochemical parameters of toxicity such as serum liver enzymes (aspartate transaminase and alanine transaminase), lactate dehydrogenase (LDH), blood urea nitrogen (BUN) and creatinine levels [carried out at Unique Bio Diagnostics Enterprises (Mumbai, India)]. Animals were then sacrificed by cervical dislocation, and the organs (liver, kidney, heart, stomach, lungs, spleen, intestine and brain) were collected directly in PBS to remove the blood traces, wrapped in aluminium foil and stored at −80°C until further use for biodistribution studies by ELISA or histology.

Tissue homogenate was prepared by taking a part of the tissue from each organ, thawed on ice, weighed in a 2-ml Eppendorf tube and extracted in extraction buffer, PBS pH 7.4 (four volume/g of organ weight). Protease Inhibitor Cocktail Set III was added to the extraction buffer (1/20th volume of the tissue weight) and subjected to homogenization using an electric homogenizer (24,25). Prior to homogenization, the probe was dipped in ice and the tissue was homogenized at 4°C with 8–10 oscillate up- and down-strokes for soft tissues derived from organs such as the liver, stomach, small intestine, lung, spleen, kidney and brain; whereas, hard tissues from the heart required more strokes and agitation. After homogenization, each sample was centrifuged for 20 min at 16,000 rpm at 4°C and the supernatant was collected and stored at −80°C until further use for analysis.

A calibration curve was constructed using different concentrations of SRL (0.002–0.312 µg/ml and 0.002–0.078 µg/ml) including a lower limit of quantification (LLOQ) for evaluating SRL in organ and serum extracts, respectively. Analytical recovery by ELISA was evaluated by adding serum and organ extracts at a 1:100 dilution and using the respective calibration curves and measuring its recovery using the same procedure and conditions as described in the ELISA experiments. The absorbance was measured at 450 nm and values were plotted against the concentrations. The LLOQ was set as the lowest measurable concentration with acceptable accuracy and precision not >20% of the expected values (26,27). Precision and accuracy were determined by analyzing quality control samples (QCs). Recovery was computed by comparing responses in triplicate of extracted QCs (28,29).

Indirect ELISA was performed using 96-well plates. The plates were pre-coated with a known concentration of SRL (0.002–0.3 µg/100 µl/well), serum samples or tissue homogenate (1:100 dilution) in coating buffer (15 mM sodium carbonate and 17 mM bicarbonate buffer, pH 9.6, containing 3 mM sodium azide) and incubated overnight at 4°C. Wells were washed thrice with 50 mM PBS, at pH 7.4 containing 0.1% (v/v) Tween-20 (PBST) to remove the unbound proteins. Non-specific binding was blocked by incubating with 100 µl of 1% BSA in PBS for 2 h at RT at 37°C, followed by washing twice with PBST to remove excess blocking agent. Wells were incubated with rabbit anti-SRL polyclonal antibodies (100 µl/well, 1:10,000 dilutions in PBS) overnight at 4°C. After washing, HRP-conjugated anti-rabbit IgG secondary antibodies (Sigma-Aldrich) at 1:10,000 dilutions were added and incubated for 2 h at RT at 37°C. The colour was developed by the addition of 3,3,5,5-tetramethylbenzidine (TMB; Sigma-Aldrich). The reaction was stopped using 4 M H₂SO₄ (100 µl/well) and the absorbance was recorded at 450 nm on a microtiter plate reader (Tecan, Crailsheim, Germany).

Histochemical examinations were performed using the tissue samples of the tumors and organs including liver, spleen, kidney, heart, lung, stomach, small intestine and brain. Excised tumours were collected and fixed in 10% formalin and embedded in paraffin blocks, and 5-µm sections were used for hematoxylin and eosin (H&E) staining and for immunohistochemistry (IHC). The histological sections were observed under an optical microscope and photographed.

Sections (5-µm thick) of the SRL-treated tumours with the controls were prepared by using the tumour tissues collected from the snap-frozen samples stored at −80°C and stained with a specific mouse anti-CD31 (PECAM-1) antibody (clone MEM-05) according to the manufacturers instructions. The primary antibody (1:500) was diluted in PBS and incubated with samples at RT for 1 h followed by washing with TBST and then with HRP-conjugated goat anti-mouse IgG (H+L) secondary antibodies (1:3,000) diluted in PBS and incubated at RT for 1 h. Finally the slides were developed using 3,3-diaminobenzidine (DAB) substrate and counterstained with hematoxylin. Images (magnification, ×10) were captured to monitor CD31 expression.

The antitumor activity (as measured by differences in tumor mass) was expressed as mean ± standard deviation (SD). In vivo data and ELISA calibration curve with unknowns was analyzed using GraphPad Prism 6. The statistical significance of differences in tumor volume was determined by one-way analysis of variance (ANOVA), followed by Sidak test for three or more groups; a value of P<0.05 was considered as significant.

Mice injected intraperitoneally with SRL (30 mg/kg body weight/mouse) showed significant inhibition (35.8%, P<0.0001) of tumour growth when compared with that observed in the control group (Fig. 1). The average initial tumour volume in the TBS and SRL-treated groups was 155±4.7 and 147±10.9 mm³, respectively before tumour cell treatment and the average tumour volume after completion of 9 doses was 353±71.2 and 171±16 mm³, respectively (P<0.001). No noticeable change in the weight and tumour volume of either control or treated group was observed after maintaining treatment for 14 days. The histology of the tumour sections of the SRL-treated and untreated animals showed marked differences. The SRL-treated mouse tumours appeared more like normal ones while the TBS-treated control mouse tumours showed a characteristic tumour appearance. The histology of liver sections showed that SRL-treated mice appeared normal compared to the control-treated mice bearing tumours, indicating the tumour-regressing effect of SRL (Fig. 2). Moreover, all the treated animals were healthy without any significant changes in body weight compared to the controls. Complete blood count (CBC) revealed no detectable changes in the SRL-treated and TBS-treated groups (Table I). PCR analysis of the tumours obtained from the experimental mice showed similar product size of 275 bp as that of the implanted HT29 cells, indicating that the tumours which developed in nude mice were from the implanted human cancer cells and not from the mice (Fig. 3).

SRL-treated animals also showed clear reduction in the number of blood vessels in the excised tumours compared with that from the control-treated animals (Fig. 1), indicating an effect of SRL treatment on suppression of angiogenesis. The anti-angiogenesis effect of SRL was also supported by the immunohistological analysis in which the expression of PECAM (CD31), an angiogenesis marker, was observed to be much lower in the SRL-treated animals than that in the control-treated animals (Fig. 2B).

Pharmacokinetic profile of SRL in Swiss albino mice was obtained by evaluation of serum samples from the SRL-treated mice by ELISA. Mice injected with SRL (24 mg/kg body weight/mouse) intraperitoneally showed maximal SRL concentrations in serum at 24 h (8.02 µg/ml) (Fig. 4A). These results indicate that SRL was not accumulated in the body and was eliminated. Biochemical parameters in serum samples collected at 24 h and analysed for hepatic function namely SGPT, SGOT, bilirubin and cholesterol exhibited no significant alterations between the SRL-treated mice and control-treated mice. Biochemical parameters related to kidney functions including urea, uric acid creatinine and total protein also showed no significant differences between the SRL-treated and control group (Table II). These results indicate the non-toxic nature of SRL treatment in the mice.

The presence of SRL in various organs including the liver, kidney, lung, spleen, stomach, small intestine, heart and brain, respectively was analysed. The lectin was detected in trace amounts in the kidney but not in any other organ (Fig. 4B). SRL at 76, 113, 79 and 22 µg/g was detected in the kidney at 1, 6, 24 and 48 h, respectively. H&E staining of tissue sections of the different organs showed no morphological changes at 24 h between the SRL-treated or control-treated animals (Fig. 5). These results suggest that SRL is effectively cleared in the body and is non-toxic to mice.