The raw material of J. chinensis was collected in Hsinchu City (GPS coordinate: 24°47'58.4"N 120°59'13.0"E). The raw material was further authenticated as J. chinensis L. var. sargentii Henry using internal transcribed spacers (ITS) of nuclear ribosomal DNA, and the ITS sequence had been deposited in the GenBank data base (Accession Number: KX496336). A voucher specimen was deposited at the plant collection bank of Industrial Technology Research Institute (ITRI,Hsinchu, Taiwan).
Preparation of CBT-143-S-F6F7 fraction from J. chinensis
Dried thin branches and leaves of J. chinensis L. var. sargentii Henry were immersed in an 8- to 10-fold weight of 95 % ethanol. Furthermore, for obtaining an extract solution, the immersed plant materials were extracted at the boiling point of the solvent for 1 h. The extract solution was then filtered using filter paper and evaporated to dryness in vacuum. The extract was further separated using a column (inner diameter: 5.5 cm; length: 30 cm), which contained a 15-fold weight of silica gel (0.040–0.063 mm, Silica gel 60, Merck), by using an acetone:n-hexane mixture (from1:4 to 1:1) as the eluent. After separation, the CBT-143-S-F6F7 fraction was identified according to the results of a tube formation assay of human umbilical vein endothelial cells (HUVECs).
Five HCC cell lines, Huh7, Hep3B, PLC/PRF/5, SK-Hep-1, and HepG2, were used. Huh7 was obtained from the Japanese Collection of Research Bioresources. Hep3B, PLC/PRF/5, and SK-Hep-1 were purchased from the American Type Culture Collection. HepG2 cells and HUVECs were obtained from the Bioresource Collection and Research Center, Taiwan. Hep3B cells were maintained in Eagle’s minimum essential medium supplemented with 10 % heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. The other HCC cells were grown in Dulbecco’s minimum essential medium supplemented with 10 % FBS, 100 mM nonessential amino acids, 100 U/mL penicillin, and 100 μg/mL streptomycin. All cells were cultured in a humidified incubator at 37 °C and 5 % CO2. All reagents for cell culture were purchased from Life Technologies (Grand Island, NY, USA).
Female BALB/c (BALB/cAnNCrlBltw) and SCID (CB17/Icr-Prkdcscid/CrlBltw) mice, aged 6–8 weeks and weighing 16–22 g, were purchased from BioLASCO Taiwan Co. Ltd. (Ilan, Taiwan). One week before the study, all mice were housed in conventional cages in the Animal Laboratory for Biomedical Research (fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care in 2011) of Industrial Technology Research Institute and allowed to acclimatize and recover from shipping-related stress. Sterilized water and food were provided ad libitum, and the mice were housed in rooms maintained at 22 °C–26 °C with 40–70 % humidity and a controlled 12-h light–dark cycle. All experimental procedures for animal studies were approved by the Institutional Animal Care and Use Committees of ITRI (subcutaneous xenograft study: ITRI-IACUC-2010-009 V1; Matrigel plug assay: ITRI-IACUC-2010-058 V1; orthotopic model: ITRI-IACUC-2011-020, and chorioallantoic membrane assay [CAM]: ITRI-IACUC-2011-007).
HUVEC tube formation assay
Tube formation assay was performed as previously described . HUVECs were cultured in growth-factor-reduced Matrigel (BD Biosciences, Franklin Lakes, NJ, USA; 356231) and treated with different CBT-143-S-F6F7 concentrations. The total length of the net structure was calculated using NIS element image analysis software (Nikon; Agent: Lin Trading Co., Ltd. Taiwan). By considering the total length of a net structure formed from a group without drug treatment as 100 %, the state of inhibition of HUVEC net structure formation elicited by different drug treatments was analyzed.
HUVEC migration assay
The ability of CBT-143-S-F6F7 to inhibit HUVEC migration was observed using a BD transwell system. HUVECs were seeded into an upper chamber in a medium without FBS, and a medium with 10 % FBS was then added to a lower chamber as an attractant. After 24 h, the transwell was collected, and the migrating cells on the lower side of membrane were stained with crystal violet to facilitate observation.
Matrigel plug assay
Matrigel (BD Biosciences, 354234) was mixed with PBS in a 9:1 ratio for mock control or 500 ng/mL FGF-b (Peprotech, USA, 100-18B) and 500 ng/mL VEGF (R&D, USA, 293-VE-500) for vehicle control. For studying antiangiogenesis, different CBT-143-S-F6F7 concentrations were added to Matrigel mixed with FGF-b and VEGF. Fourteen days after subcutaneous Matrigel injection, BALB/c mice were sacrificed, Matrigel plugs were collected, and hemoglobin in the plugs was quantified using a QuantiChrom Hemoglobin Assay Kit (BioAssay Systems, USA, DIHB-250).
The embryos of unhatched eggs from specific-pathogen-free white Leghorn chicken were placed laterally in an incubator (37 °C, relative humidity: 55–60 %). On day 4 of the incubation period, 2.5 mL of albumin was drawn from the eggs using a 20-G needle, and a fake air chamber was constructed on the embryo. Subsequently, the needle hole and fake air chamber were sealed using 3 M breathable tape. On day 7, CBT-143-S-F6F7 was dissolved with dimethyl sulfoxide (DMSO) and diluted with PBS. For each group, including a vehicle group, the final DMSO concentration was 1 %. CBT-143-S-F6F7 was loaded on 6-mm-diameter Circular Advantec filter paper (Toyo Roshi Kaisha, Ltd., Tokyo, Japan), and the paper was placed on the CAM. On day 9, the CAM was photographed using the SZX16 dissecting microscope (Olympus, Japan). By using the filter paper as the center, four concentric circles were marked on the photograph (diameters of 7, 8, 9, and 10 mm; total circumference of the four circles was 106.8 mm, and total circumference represented the region near the filter paper) . The amount of vessels crossing the concentric circles (the vascular density index or VDI) was used to evaluate the state of angiogenesis. The VDI for the same photograph was determined by three people, and the mean VDI was adopted.
Cell viability assay
HCC cells were seeded in 96-well plates at 104 cells/well and treated with CBT-143-S-F6F7 for 48 h. The cells were then exposed to a culture medium containing 0.5 mg/mL of (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) (Sigma–Aldrich, St. Louis, MO, USA). After 2–3 h, DMSO was added to dissolve the resulting formazan. For each solution, the optical density at 570 nm was measured using a microplate spectrophotometer (SpectraMax M5, Molecular Devices, USA). Cell viability was calculated as follows: viability (%) = A570 of treated cells/A570 of control cells × 100. The half maximal inhibitory concentration (IC50) was estimated using GraFit software.
In vivo subcutaneous tumor model
Huh7 cells (3 × 106) were suspended in 100 μL of PBS with 25 % Matrigel and subcutaneously implanted into the right flank of female SCID mice. Tumors were measured three times per week by using calipers, and tumor volume was calculated as follows: tumor volume (V) = (LS2)/2 (L, longest diameter, mm; S, shortest diameter, mm). Treatments were initiated when the tumor volume reached 150–200 mm3. The mice were randomly grouped into groups of six. CBT-143-S-F6F7 was dissolved in a formulation of 10 % 1-Methyl-2-pyrrolidone (M6762, Sigma–Aldrich), 20 % Cremophor EL (C5135, Sigma–Aldrich), and 70 % saline. CBT-143-S-F6F7 was administered daily through gavage at a dose of 100 mg/kg body weight for 21 days. The tumor volume and body weight of the mice were monitored. The antitumor activity of treatments was represented by the percentage of tumor growth inhibition (TGI), which was calculated as follows: [1 − (final tumor volume – initial tumor volume for treated group)/(final tumor volume – initial tumor volume for vehicle group)] × 100. Data are represented as the mean ± standard error of the mean (SEM).
In vivo orthotopic tumor model
Mice were anesthetized with a mixed solution [40 mg/kg body weight of Zoletil 50 (Virbac, France) and 10 mg/kg body weight of Xylazine (Rompun 2 % injection, Bayer, German)] through intraperitoneal (i.p.) injection. Through an upper middle abdominal cavity incision, the left lateral lobe of the liver was exposed. Huh7 cells (1 × 106) were suspended in 50 μL of PBS with 50 % Matrigel and were implanted into the left lateral lobe of the liver by using an insulin syringe with a needle (29 G X1/2", Terumo, Shibuya, Japan), and the wound was then closed using an interrupted suture and Autoclip wound closing system (59043, Stoelting, USA). Seven days after the implantation, CBT-143-S-F6F7 treatment (100 mg/kg through oral administration) was initiated and proceeded for 42 days. The body weight of the mice was monitored twice a week, and blood samples were collected from the facial vein. Survival analysis was conducted using the Kaplan–Meier method and GraphPad Prism® was used for generating survival curve and performing related statistics. The human α-fetoprotein (AFP) level in mouse serum was determined using AFP ELISA (IB19102, IBL-America, USA).
After the mice were sacrificed, tumor tissues were fixed in formalin and embedded in paraffin as tumor sections for further analysis. Immunostaining was completed using an Autostainer Link 48 system (Dako, Glostrup, Denmark). The antihuman Ki-67 (IS-626, Dako), antihuman cyclin D1 (1:500, IS-083, Dako), and anti-CD31 antibodies (LS-B1932, Lifespan, USA) were used for immunostaining. Five fields (100 × 100 μm2) of each tumor sample were randomly selected, and the percentages of Ki-67-positive and cyclin D1-positive cells were calculated to evaluate cell proliferation in tumor tissues. For quantifying CD31-positive cells, ten fields of each tumor sample were randomly selected. The results are presented as the mean ± standard deviation (SD).
Flow cytometry for cell cycle analysis and annexin V staining
For cell cycle analysis, Huh7 cells were seeded in 6-well plates at 2 × 105 cells/well and treated with CBT-143-S-F6F7 for 48 h. The cells were collected through trypsinization and fixed overnight in 70 % ethanol at −20 °C. The fixed cells were then washed with PBS. For measuring DNA content, the cells were stained with a propidium iodide (PI) solution [2 μg/mL PI, 200 μg/mL RNase A, and 0.1 % (w/v) Triton X-100] (Sigma–Aldrich) and incubated in the dark for 30 min at 37 °C. DNA content was determined using a FACScan flow cytometer (BD Biosciences). Data were analyzed using ModFit LT software (Verity Software House, USA).
For apoptosis analysis by using PI and annexin V staining, Huh7 cells were seeded in 6-well plates at 2 × 105 cells/well and treated with CBT-143-S-F6F7 for 48 h. The cells were collected through trypsinization, and PI and annexin V staining was then conducted using an annexin V–fluorescein isothiocyanate apoptosis detection kit (Strong Biotech Corp., Taipei, Taiwan).
Protein array analysis
Huh7 cells were treated with CBT-143-S-F6F7 for 48 h, and the cell lysate and condition medium were collected and analyzed using the Proteome Profiler Human Apoptosis Array Kit (R&D, ARY009) and Proteome Profiler Human Angiogenesis Array Kit (R&D, ARY007), respectively. The operating procedures were performed according to instructions in the user manual. Chemoluminescent signals were monitored using the BioSpectrum 610 imaging system (UVP, USA) and analyzed using ImageJ software.
Identification of active compounds from CBT-143-S-F6F7
CBT-143-S-F6F7 was further purified using a fast protein liquid chromatography (FPLC) system, which was conducted using GE Healthcare Unicorn 5.1 controller equipped with P-900 pump, superloop 50 mL, UV-900 monitor detector and Frac-920 fraction collector. FPLC was performed with Toyo Pearl® HW-40S column (inner diameter: 3 cm; length: 30 cm; flow rate: 5 mL/min) and then eluted with 95 % ethanol to yield five fractions. Fraction 2 was further purified using a column (inner diameter: 2 cm; length: 20 cm) packed with 100 g silica gel (0.015-0.040 mm, Silica gel 60, Merck) and then eluted with Ethyl acetate/n-hexane (1:5) at flow rate of 10 mL/min to yield deoxypodophyllotoxin, acetyl podophyllotoxin and yatein. Fraction 3 was further purified using a column (inner diameter: 2 cm; length: 25 cm) packed with 120 g silica gel (0.015-0.040 mm, Silica gel 60, Merck) and eluted with ethyl acetate/n-hexane (1:3) at flow rate of 10 mL/min to get savinin. Fraction 4 was further purified using a column (inner diameter: 2 cm; length: 25 cm) packed with 120 g silica gel (0.015-0.040 mm, Silica gel 60, Merck) and then eluted with ethyl acetate/n-hexane (1:3) at flow rate of 10 mL/min to obtain oxohinokinin. The structures of these compounds were identified using nuclear magnetic resonance (NMR) analysis. 1H (600 MHz) and 13C (150 MHz) NMR spectra, DEPT, 1H-1H COSY, NOESY, HSQC, and HMBC experiments were recorded by a varian unity inova 600 NMR spectrometer at room temperature. Chemical shifts were reported in δ units and coupling constants (J) in Hz.
To determine the content of oxohinokinin, savinin, deoxypodophyllotoxin, acetyl podophyllotoxin, and yatein in CBT-143-S-F6F7, high performance liquid chromatography (HPLC) was conducted by using a Waters system (Waters 600E controller and Waters 717 plus autosampler) equipped with 996 photodiode array (PDA) detector (Waters, 2998). HPLC was performed by using a Cosmosil 5C18-MS-II C-18 reversed phase column (250 mm x 4.6 mm, 5 μm diameter particles) and the flow rate is 0.8 mL/min. Eluents in reservoirs A-C were as follows: 0.1 % phosphoric acid in water (A), acetonitrile (B), methanol (C). Delivery programs f linear gradient are A/B/C = 65/25/10 to A/B/C = 25/60/15 for 60 min. UV absorption spectra were recorded at 280 nm.
All experimental data are expressed as the mean ± SD, unless otherwise stated. The significance of differences between groups was analyzed using the Student’s t test. P < 0.05 was considered statistically significant.