2.1 Cell culture conditions
Human NSCLC cells including H1975 (wild-type p53, two mutations in EGFR (L858R/T790M, erlotinib-insensitive)), H3255 (mutant p53, one
mutation in EGFR L858R, erlotinib-sensitive), A549, H1299 and H460 were acquired from American Type Culture Collection (Manassas, VA) and cultured in 75 cm2 tissue culture flasks. The cells were grown in RPMI (Roswell Park Memorial Institute medium) or DMEM with supplementation of 10% FBS (Thermo Fisher Scientific, Waltham, MA), 100 unit/mL penicillin and 100 μg/mL streptomycin and cultured at 37°C in the environment of humidified incubator with 5% CO2. The cell media was replaced every three or four days and sub-cultured. Cells reaching 80-90% confluence were used for experiments.
2.2 The spheroid culture
Spheroid cultures were initiated by seeding a total of 1 × 106 cells in 4 mL of medium per 60 mm petri dish pre-coated with poly-HEMA. This
provides a coat of sufficient homogeneity to completely inhibit cell attachment.
The cultures were kept at 37℃ in a humidified CO2 incubator with weekly change of 50% of the medium. Spheroid bodies were derived by placing the parental cells into DMEM/F12 (DMEM Nutrient Mixture F-12) culture medium containing 2% B-27 supplement, human bFGF (10 ng/ml) and EGF (20 ng/ml, PEPROTECH).
2.3 Chemicals and reagents
Sun Ten Pharmaceutical (Taichung, Taiwan) provided the aqueous extracts of the whole plant following the published procedures. Briefly, the collected materials samples were mixed with sterile water before boiling. The supernatant following centrifugation was filtered, concentrated and adjusted to a final concentration of 1 g/mL before storage.
The chemicals including PI, RNase A, trypan blue, Tris-HCl and Triton X-100, were from Sigma-Aldrich Chemical (St. Louis, MO) and penicillin–
streptomycin, glutamine, trypsin-EDTA and DMEM medium (Thermo Fisher Scientific).
2.4 Liquid chromatography/mass spectrometry analysis and instrumental conditions
The LC/MS (Liquid chromatography/mass spectrometry) method was used to identify the major markers of bioactive substances. The system for analysis consisted of a LC-20AD UFLC system (Shimdzu, Kyoto, Japan) linked to a LCMS-8040 triple quadrupole mass spectrometer. The running condition was designed as follows: gradient elution by the mixture of mobile phases A
(0.1% formic acid and 1 g/L solution of ammonium acetate in water) and B (0.1%
formic acid and 1 g/L solution of ammonium acetate in methanol) at minutes 0–
40 with the ratio of 100%–70% in A and 0%–30% in B; at minutes 40–70 with the ratio of 70%–0% in A and 30%–100% in B; at minutes 70–70.1 with the ratio of 0%–100% in A and 100%–0% in B; and at minutes 70.1–80 with the ratio of 100% in A and 0% in B. The flow rate was fixed at 0.4 mL/min and column temperature kept at 40°C. The injection volume was adjusted at 30 μL and the analytical column used a Shim-pack XR-ODS II column (2.2 μm,
2×100 mm, Shimadzu). Dual ion modes (ESI (+) and (-)) were used in MS detection and the transmission of (M+H)+ and (M-H)- was set as the optimum condition. The MS detection was arranged as full scan range (400–800 amu).
The interface voltages were set at 4.5 kV for ESI (electrospray ionization) (+) and -3.5 kV for ESI (-). With nitrogen as nebulizing and drying gas, the flow was set at 3.0 and 10 L/min, respectively. Argon as collision-induced
dissociation gas was kept at 230 kPa. Desolvation lines temperature was set at 150°C and heat block temperature maintained at 400°C.
2.5 Protein extraction and western blot analyses
Human NSCLC sphere were plated (1 × 106 cells/well), cultured in above-mentioned spheroid culture conditions, and then treated for an additional 12 h with aqueous BJ (5, 10 and 15 mg/ml). Spheres collected by pipetting were lysed by RIPA (Radioimmunoprecipitation assay) buffer (1 % Triton X-100, 150 mM NaCl, 5 mM EDTA, 20 mM sodium phosphate and protease inhibitor
cocktail for use with mammalian cell and tissue extracts (Sigma-Aldrich)).
Samples were heated for 5 min at 95°C. Lysates from cells were
electrophoresed in 10% polyacrylamide gel. The proteins in the gel were transferred to nitrocellulose blotting membrane. The membrane was saturated for 1 h with 5% non-fat dry milk in Tris-buffered saline (pH 7.6). The blots were then incubated with apoptosis pathway antibodies caspase-3, PARP (Genetex) and EGFR, pEGFRY1068 (Genetex). The spheroid protein was incubated with CD133, ALDH1A1 (aldehyde dehydrogenase) and Nanog (Genetex) and then was exposed to secondary antibodies and visualized by LAS-4000 (FUJIFILM). Molecular weights are determined by comparison to
2.6 Soft-agar colony formation assay
Anchorage-independent growth was determined in agarose (0.33%), as described [30]. Spheroids were plated in a semisolid medium (DMEM,
supplemented with 10% FBS and 0.5% agar). Dishes were incubated at 37°C in a humidified atmosphere containing 5% CO2 and colonies were stained by 0.002% crystal violet and counted after 25-28 days.
2.7 Immunofluorescence Staining
Spheroids were cultured under the spheroid conditions for 7-10 days.
Spheroids were fixed in 4% paraformaldehyde and incubated with primary antibody for two days at and with secondary antibody overnight at 4°C. The primary antibodies were used include Nanog, ALDH1A1 and BrdU (Genetex).
2.8 BrdU incorporation of spheroids
The assays to measure the proliferation of spheres were BrdU Labeling and Detection Kit II (Roche, Mannheim, Germany). The enriched spheroids treated with various concentrations of BJ for 12 h were incubated in media containing 10 μM BrdU for 1 h. Fixatives of 3:7 mixture of 50 mM glycine solution (pH 2.0) and ethanol were added, followed by incubation overnight at -20°C. The slides were washed with PBS and incubated at 37°C in solution containing diluted anti-BrdU antibody (1:1,000 dilution) for 30 min. The cells were suspended in a mixture of 1% BSA containing Alexa Fluor 488-conjugated secondary antibody (1:250 dilutions) for 2 h in darkness. An inverted
fluorescence microscope was used to record the fluorescent images. All colored images were converted to black and white by Photoshop software before being
quantitated with Multi Gauge software (version 2.1, FUJIFILM). The green fluorescence at each concentration was obtained and compared with that of water control as BrdU intensity ratio. Three independent experiments were carried out.
2.9 Tumor xenograft study
A subcutaneous xenograft animal model was used to evaluate
tumorigenesis variations. The female nu/nu mice of three- or four-week old were obtained from National Applied Research Laboratories (Taipei, Taiwan).
The animals were housed under aseptic and ventilated conditions free of pathogens with a 12-hour light-dark cycle. The study was approved by the Animal Committee of National Taiwan Normal University, Taipei, Taiwan.
Firstly, a total of 1 × 106 exponentially growing H1975 cells were seed on poly HEMA coated dish under the stem cell culture conditions as above mentioned.
After 5-7days later, a total of 8 × 104 H1975 cancer spheroid cells in 0.2 mL (1:1 mixture ratio of PBS and 100 μL Matrixgel™ Basement Membrane Matrix (BD Biosciences) were subcutaneously injected into the dorsal area of nude mice. Each group consists of two or three mice. When the xenograft tumors reached 50-100 mm3 in size following cell inoculation, the mice were given an injection of 2 and 4 g/kg of BJ or vehicle control every other day to each group of mice for six days in a row. The volumes of tumors that formed were
measured by a caliper and calculated according to the equation: volume in mm3
= (A × B2)/2, where values of A and B corresponded to the largest and the smallest diameters of the implanted tumor, respectively.
Mice were anesthetized by ether and tumors were subsequently removed and fixed by 5% (w/v) formaldehyde for overnight in the refrigerator.
Formaldehyde is always freshly prepared from paraformaldehyde just before use. For sectioning (tissue freezing methods for cryostat sectioning), firstly
tumor was sliced into 5 to 8 mm thick slabs (Cryostat for frozen section, LEICA, CM0350S). And then the slabs were washed several times in PBS (pH 7.4), passed through a series of increasing concentrations (w/v) of sucrose (10%, 20%, and 30%) in phosphate-buffered saline. The slabs were frozen,
equilibrated in a cryostat at -20°C and then sectioned serially at thicknesses of 10 μm. For immunostaining, tumor sections were incubated overnight in the presence of 5% FBS in PBS with 0.2% Triton X-100. The tumor sections were washed several times in PBS, then incubated primary and secondary antibodies.
2.10 TUNEL assay
The apoptotic cell death was evaluated by TUNEL staining. Spheroids were treated with different concentrations of BJ extract for 12 h were
permeabilized in 10 mM citrate buffer, pH 6.0. After blocking the nonspecific labeling with PBS mixture containing 2% BSA and 0.5% NP-40, cells were incubated in TUNEL reaction solution mixed with 9 mM dUTP, 1 mM digoxigenin-labelled dUTP (Roche, Mannheim, Germany), 2.5 mM cobalt chloride, 100 mM Tris pH 7.6 and 0.3 U/l terminal deoxynucleotidyl transferase for 1 h at 37°C in a humidified atmosphere. The spheroids were washed with PBS and incubated with a 1 : 200 dilution of horseradish peroxidase-conjugated digoxigenin antibody (Roche). With removal of unbound antibody, cell images were taken by fluorescence or confocal microscope. All colored TUNEL images were converted to black and white ones by Photoshop software before being quantitated with MultiGauge software (version 2.1, FUJIFILM). Five slides of
each concentration and control sections were recorded and three independent experiments carried out.
To identify apoptotic cells in vivo, tumor sections (10 μm) were stained with a TUNEL assay kit (Roche) according to the manufacturer’s instructions.
Under the fluorescence microscope, the apoptotic cells were exhibited green fluorescence in the nucleus after excitation with blue light. Quantitative number of TUNEL-positive cells was counted total positive cells in 100 H1975 tumor cells at each concentration. Five slides of each concentration and control sections were recorded and three independent experiments carried out.
2.11 PCNA antibody staining for marker of proliferating cells in vivo To determine proliferating cells in vivo, tumor sections were incubated with anti-PCNA (proliferating cell nuclear antigen) antibody. Under the
fluorescence microscope, the proliferating cells exhibited green fluorescence in the nucleus after excitation with blue light. Quantitative number of PCNA- positive cells was counted total positive cells in 100 H1975 tumor cells at each concentration.
2.12 Statistical analysis
The student’s t-test was applied to evaluate the differences between treated and control animal groups. The statistical analysis was performed by paired Student’s t-test using SigmaPlot 12.0 software. All values were presented as mean ± SE (Standard errors) unless otherwise noted. For all the tests, the level of significance was set at *p<0.05 and **p<0.01.