Materials
The Pinellia ternata purchased from Jinmen Hubei province had been identified for its authenticity by Keli Chen in Chinese Medicine of Hubei College of traditional Chinese Medicine; PHE Sepharose Cl-4B and DEAE Sepharose Fast Flow were purchased from Amersham Pharmacia Biotech; Roswell Park Memorial Institute 1640 (RPMI 1640) was from HyClone (USA); CTX was purchased from Wuhan University Zhongnan Hospital; MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and Dulbecco’s Modified Eagle’s Medium (DMEM) were products of Sino-American Biotechnology Company. Kunming Mice were purchased from Laboratory Animal Center of School of Medicine in Wuhan University and the experiments in vivo were performed in the same center. Mice blood was obtained by puncturing the marginal ear veins of healthy animals. The Sarcoma 180 and Hela cells were obtained from China Center for Typical Culture Collection. All other chemicals used were analytical grade reagents unless otherwise mentioned.
Extraction and purification of lectin from the bulbs of Pinellia ternata
The bulbs of Pinellia ternata (100 g) were homogenized for 30 s at the internal interval of 1 min in deionized water at 4°C for 4 h. The extract was centrifuged at 8000 rpm for 40 min. The resultant pellet was discarded and the proteins in the supernatant were brought to 40% saturation of ammonium sulphate by stirring slowly, cooled at 4°C overnight and then centrifuged at 8000 rpm for 40 min. The precipitate was dissolved in 20 mM Tris–HCl buffer (pH 7.4) and dialyzed against the same buffer containing 1.5 M ammonium sulphate. After 16 h of dialysis, the sample was applied onto PHE Sepharose Cl-4B column which had been pre-equilibrated with “Tris–HCl buffer” mentioned above. Elution was carried out with the gradient buffer from “Tris–HCl buffer” containing 1.5 M ammonium sulphate to “Tris–HCl buffer” containing 0 M ammonium sulphate and monitored at 280 nm. The fractions exhibiting hemagglutinating activity were collected, dialysed against “Tris–HCl buffer” and then concentrated by centricon (PM-3). The concentrated active parts were loaded to a DEAE-Sepharose Fast Flow column (25 mm × 160 mm), which had been pre-equilibrated with “Tris–HCl buffer”. The bound protein was eluted by a continuous linear gradient of “Tris–HCl buffer” containing 0–0.3 M NaCl. The fractions exhibiting hemagglutinating activity were pooled and used for further studies. The purified lectin was designated as PTL.
Protein concentration assay, hemagglutination assay and carbohydrate analysis
We measured the soluble protein content with the Bradford assay (Bradford [1976]) using bovine serum albumin as a standard. Hemagglutinating activity was measured in V-well microtitre plates (Cao et al. [2010]). A total volume of 0.1 ml was used in each well: 10 μl aliquots of serial three-fold dilutions of PTL in PBS, 20 μl of 2% suspension of mice erythrocytes in PBS and 70 μl of PBS were incubated for 2–3 h at room temperature and titre of visible agglutination by eye-sight was noted. One hemagglutination unit is defined as the lowest concentration of lectin that causes visible erythrocyte agglutination. The positive and the negative control were Concanavalin A and PBS respectively. Carbohydrate content of the purified lectin was determined by the phenol sulphuric acid method as described by the Dubois method using D-glucose as a standard (Dubois et al. [1956]).
Homogeneity and molecular mass determination
SDS-PAGE was performed using 12.5% (w/v) acrylamide in gels, the molecular weight standard was the low molecular weight markers: beta-galactosidase (E. coli) (116.0 kDa), BSA (66.2 kDa), ovalbumin (45.0 kDa), lactate dehydrogenase (35.0 kDa), restriction endozyme (25.0 kDa), beta-lactoglobulin (18.4 kDa) and a-lactalbumin (14.4 kDa).
Native molecular mass of the purified lectin was detected by Sephacryl S-100 column (2.0 cm × 75 cm) which had been pre-equilibrated with 20 mM PBS (pH 7.4). Elution was carried out with the same buffer at a flow rate of 0.4 ml/min and monitored at 280 nm. The molecular mass of the eluting lectin was estimated from a plot of the log of the molecular weight, while a distribution coefficient (Kav) was calculated from the elution volume of the standard markers. The molecular mass standards used for calibration were BSA (66.2 kDa), Galanthus nivalis agglutinin (48 kDa), soybean trypsin inhibitor (30.2 kDa), trypsin (23.3 kDa) and cytochrome C (12.5 kDa).
Mass spectra were performed by using a Voyager-DE-STR MALDI-TOF mass spectrometer. The samples were pooled and redissolved in water containing 0.1% trifluoroacetic acid for desalting with C18 ziptips before being analyzed with MS.
Amino acid composition and N-terminal determination
The lectin was hydrolyzed with 6 M HCl at 100°C for 30 h. The hydrolyzate residue in the supernatant was quickly derivatized with 9-fluorenylmethoxycarbonyl chloride and O-phthalaldehyde. The amino acid derivatives were analyzed by using HPLC with a Hypersil ODS C18 (4.6 mm × 150 mm) column. The N-terminal aa sequence analysis was performed by using an Applied Biosystems protein sequencer through automated Edman degradation.
Temperature and pH profile
To study pH stability of the lectin, purified lectin was incubated at room temperature (24°C) in 20 mg/ml of different pH buffers ranging from pH 3 to 12: NaH2PO4-citric acid buffer (pH 3.0-5.5), sodium phosphate buffer (pH 6.0-7.5), Tris–HCl buffer (pH 8.0-9.0) and glycine-NaOH buffer (pH 9.5-12). After incubation for 30 min, the residual hemagglutinating activity was calculated.
To study the effect of temperature on hemagglutinating activity, purified lectin was incubated for 30 min at 20, 30, 40, 50, 60, 70, 80, 85, 90 and 95°C in PBS (pH 7.4). After incubation, aliquots were rapidly cooled on ice and the residual hemagglutinating activity was checked. The results were expressed as percentage of residual activity relative to the control.
MTT colorimetric assay
Sarcoma 180 cell line, Human cervical carcinoma cell line (HeLa) and human leukaemia K562 cell line were used in the MTT colorimetric assay. Culturing and maintenance of these cells were followed as Yan (Yan et al. [2009]) and cell proliferation was checked by an MTT assay. Cells (1 × 105) in their exponential growth phase were seeded into each well (200 μl media per well) of a 96 well plate and incubated at 37°C in a 5% CO2 incubator. After 12 h (the cell density was about 80%), cell culture were removed and new culture was added, different concentrations of PTL (pre-sterilised with 0.22 μm film filtration) ranging from 0–40 μg/ml were added to the well and the cells were further grown at 37°C for 48 h. After removing the supernatant, 200 μl of PBS containing 5 mg/ml MTT was added and incubated at 37°C for 4 h. The supernatant was removed again and 150 μl of dimethyl sulphoxide was added into each well to dissolve the MTT formazan at the bottom of the wells. After 10 min, the absorbance was read at 492 nm using enzyme-linked immunosorbent assay plate reader. The proliferation inhibition ratio was calculated using the following equation: Proliferation inhibition ratio (%) = [(U-T)/U] × 100%; where, U is the OD492 nm of the cells without PTL treatment (control) and T is the OD492 nm of the cells with PTL treatment. All experiments were carried out in triplicate.
Establishment of sarcoma 180 tumors and experimental therapy in vivo
Sarcoma 180 cells were inoculated subcutaneously into male Kunming strain mice to establish tumors and this animal model of tumor was used to determine the antineoplastic effect of PTL on tumor growth. Kunming mouse has been widely used in medical experiments for its adaption to a variety of experimental environment. Sarcoma 180 cells, which were suspended in normal saline solution at 2.5 × 106 cells/ml, were implanted subcutaneously on right front leg armpit (Kuznetsova et al. [1999]). Each mouse received 0.2 ml mixture of tumor suspension subcutaneously. After 24 h, PTL was maintained at the different concentrations ranging from 0–3.25 mg/kg body weight for intraperitoneal injection at every other day. CTX was given at 20 mg/kg body weight to the positive control group and 0.2 ml PBS solution was given to the negative control group. On the 11th day, after the mice were sacrificed, tumors and spleens indexes were extirpated and weighed. Later, the inhibition ratio of the tumors and the organ indexes were calculated as described by Chen (Chen et al. [2003]).
Flow cytometry analysis
Sarcoma 180 cells were cultured with the methods described above and treated with different concentrations of PTL ranging from 0–40 μg/ml for 12 h and 24 h. The cells were processed by trypsin, collected and washed twice with ice-cold PBS, and then suspended in 75% ethanol at −20°C overnight. Fixed cells were centrifuged at 600 g for 5 min and washed with ice-cold PBS again. To study the DNA content and the cell cycle, cells were incubated with 100 μg/ml RNase A in PBS at 37°C for 1 h before stained in the dark with PI (20 μg/ml) at 4°C for 30 min. Samples were applied to a flow cytometer. For each analysis, a minimum of 10,000 cells were counted.
