Poncirus trifoliate (L.)Raf, Akebia Trifoliate Koidz, Citrus medica var. sarcodactylis Swingle and Saussurea lappa were purchased from Shanghai Pharmaceutical Co., Shanghai, China, and were authenticated by Prof. Bo-Wen Qian, Department of Traditional Chinese Medicine, Shanghai University of Chinese Traditional Medicine, Shanghai, China. We also used the following materials: Concanavalin A (ConA), lipopolysaccharide (LPS), propidium iodide, ADP (Sigma, St Louis, MO); [3H]TdR (China Institute of Atomic Energy, Beijing, China); human recombinant interleukin-2 (rhIL-2) (Boehringer Mannhein GmbH, Germany); human recombinant tumor necrosis factor (TNF) (Collaborative Biomedical Products, Bedford, MA); FITC-conjugated antibodies to CD3, CD4, CD8 (Pharmingen, Becton Dickinson, Franklin Lakes, NJ); lactic dehydrogenase (LDH) Cytotox assay kit (Promega, Madison, WI); 125I-TXB2 and 125I-Keto-PGF1α radioimmunoassays (RIA kits, Suzhou Medical College, Suzhou, China); RPMI Medium 1640 (Invitrogen, Carlsabad, CA); fetal bovine serum (FBS, HyClone Corporation, Logan, UT).
Mouse sarcoma 180 tumor cells, Lewis lung carcinoma cells, and human gastric carcinoma cell line SGC-7901 were donated by Shanghai Institute of Materia Medica, Chinese Academy of Sciences (Shanghai, China). YAC-1, CTLL-2, and L929 cells were provided by School of Medicine, Shanghai Jiao Tong University (Shanghai, China), and were grown routinely in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS). The medium was supplemented with 100 U/mL penicillin and 100 U/mL streptomycin; 10 ng/mL rhIL-2 was added in the culture medium of CTLL-2 cells, and the cells were incubated in a humidified atmosphere, with 5% CO2 in air at 37°C.
Male BALB/c mice and C57BL/6 mice (6 to 8 weeks old) were provided by the Department of Shanghai Institute of Planned Parenthood Research, Fudan University, Shanghai, China. The animals were group-housed in a regulated environment (22 ± 1°C, relative humidity 60 ± 5%) with a 12-h light and 12-h dark cycle (08:00–20:00, light). Food and water were given ad libitum. Food pellets meet Feed Standard of Medical Laboratory Animal of China. Food composition is as follows: protein18–25%, fat 4–5%, calcium 1.0–1.8%, phosphonium0.6–1.2%, vitamins A 12500–15000 IU/kg, vitamins D 1250–1500 IU/kg, fiber 4–5% and moisture 8–10%, lysine 0.98–1.42%, cystine0.76–1.10%, tryptophane 0.22–0.34%.
BALB/c strain athymic nu/nu mice were provided by Shanghai Cancer Institute, China. Female mice (6 to 8 weeks old) were used in this study. The animals were maintained in a specific pathogen free animal care facility, under controlled conditions (25 ± 2°C, 50%–60% relative humidity and 12-hour light cycle). They were fed with autoclaved tap water and food ad libitum. The laboratory animal protocol used for this study was approved by the committee for control and supervision of experimental animals of Shanghai University of Chinese Traditional Medicine.
Preparation of Liqi
Liqi was prepared as a lyophilized powder of hot water extracts from 4 species of medical herbs: Citrus medica var. sarcodactylis Swingle, Akebia Trifoliate Koidz, Poncirus trifoliata(L.)Raf and Saussurea lappa. Briefly, the above materials were mixed in a ratio of 3:2:1.7:1.7 and decocted three times with boiling distilled water for 1 h. The decoction was filtered, collected, concentrated, and lyophilized. The yield of liqi was approximately 20%. Liqi was dissolved in distilled water and administered in a volume of 10 ml/kg. In this experiment, the animal dose of liqi was dose of crude drug.
Tumor models and in vivo treatment regimen
Sarcoma 180 tumor cells were subcutaneously implanted (2 × 106 S180 tumor cells suspended in 50 μl of Ca2+- and Mg2+-free phosphate-buffered saline [PBS]) in the right axillary region of syngeneic BALB/c mice. Lewis lung carcinoma cells were subcutaneously implanted (2 × 106 LLC tumor cells suspended in 50 μl of PBS) in the right axillary region of syngeneic C57BL/6 mice. SGC-7901 cells were subcutaneously implanted (2×105 cells suspended in 0.2 ml of PBS) in the right axillary region of BALB/c nude mice. After 24 h, they were weighed and randomized into 5 groups (12 mice each): a normal control group, a group that was injected with tumor cells but treated with water only, and three groups that were injected with tumor cells and treated with different doses of liqi (12.5, 25 and 50 g/kg). Treatments were administrated at about 9 o'clock daily by gavage, and mice were weighted daily.
On day 21, all mice were weighed and euthanized, and tumors were removed and weighed. The tumor repression rate was calculated as follows: The repression rate (%) = (mean weight of tumors of water-treated mice – mean weight of tumors of liqi-treated mice)/mean weight of tumors of water-treated mice × 100% .
Cell cycle analysis of SGC-7901 cells from xenografted mice
For cell cycle analysis, cells were harvested from xenografted BALB/c nude mice. On day 21, tumor tissues were immediately removed and disaggregated in PBS and filtered through a double layer of stainless-steel mesh using a syringe plunger to obtain single cell suspension. Cell cycle distribution was evaluated by propidium iodide staining of nuclei and flow cytometric analysis . Briefly, pelleted cells were washed twice with PBS and then fixed in 70% cold ethanol overnight at -20°C. After washing again, the cells were resuspended in PBS containing RNase A (200 μg/mL) and incubated at 37°C for 30 min. Propidium iodide was added to the cell suspensions at a final concentration of 100 μg/mL. The fluorescence intensity of PI was analyzed with a FACS calibur flow cytometer (FACS Calibur; Becton Dickinson, USA) and Cell Quest software.
Flow cytometry for peripheral blood T lymphocyte subsets
On day 12, the heparinized peripheral blood was collected from the orbital plexuses of the tumor-bearing C57BL/6 mice to analyze peripheral blood lymphocyte subsets by flow cytometry. Samples were prepared by adding 5 μl of fluorescent monoclonal antibodies against CD3, CD4, and CD8 to 100 μl of heparinized whole blood. CD3+CD4+ (T helper cells) and CD3+CD8+ (T suppressor cells) were counted. Lymphocytes were separated from erythrocytes lysed. The tubes were placed on ice in the dark until flow cytometric analysis; five thousand cells were collected for each sample, and data were analyzed by using flow cytometry.
IL-2 activity assay
In order to determine the effects of Liqi on IL-2 production and activity, splenic lymphocytes from tumor bearing BALB/c mice were prepared after treatment with liqi (50 g/kg) for 12 days as described above. Spleens of mice were removed under sterile conditions and disaggregated in PBS and filtered through a double layer of stainless-steel mesh using a syringe plunger to obtain single-cell suspension. Lymphocytes were collected and suspended in RPMI-1640 medium at a concentration of 1×107 cells/mL, with ConA added to a final concentration of 5 μg/mL. After incubating at 37°C and 5% CO2 for 24 hours, cells supernatants containing extracellular IL-2 were collected and stored at -20°C until assay.
IL-2 activity was tested using an IL-2-dependent CTLL-2 cell proliferation assay . The CTLL-2 cells were washed in medium containing 2% FBS and incubated for 2 h without IL-2. The cells were then cultured (1× 105 cells/mL) in 96-well plates with 0.6 ng/mL of rhIL-2 or with spleen cell supernatant for 24 h. Six hours before the end, 0.5 μCi [3H]TdR 50 μL was added to each well. The [3H]TdR incorporation was measured with a liquid-scintillator (Beckman Co, USA) counting technique. The results were described as the average of triplicate Bq (specific radioactivity of [3H]TdR was 20 μCi/mmol).
Evaluation of NK cell cytotoxicity
NK cells from tumor bearing BALB/c mice spleens were prepared as described above and used as effector cells. YAC-1 cells, mouse lymphoma sensitive to NK cells were used as target cells. Effector and target cells resuspended in RPMI-1640 medium were added to each well of a 96-well round-bottom microculture plate in triplicate to obtain an effector/target (E/T) ratio of 100:1 and incubated for 16 h. The amount of released lactate dehydrogenase (LDH) in culture supernatants was determined using the LDH Cytotox assay kit according to the manufacturer's instructions. The OD was read at 490 nm with a Microplate Reader. The percentage of NK cell cytotoxicity was calculated with the formula: cytotoxicity (%) = (experimental release – effector spontaneous release – target spontaneous release)/(target maximum release – target spontaneous release) × 100. Experimental release stood for LDH that was released from cocultures at an E/T ratio of 100:1, effector spontaneous release or target spontaneous release was spontaneous LDH release from effector or target cells incubated with medium alone, and target maximum release was obtained from target cells lysed with the lysis solution .
Production and activity assay of TNF
On day 12, tumor-bearing BALB/c mice received 20 μg/0.2 ml of LPS intravenously. After 1.5 h, blood samples were collected from the orbital plexuses of the mice. The sera were stored at -20°C until used for the TNF assay. TNF activity was determined by a cytotoxicity assay on L929 cells . Briefly, L929 cells were plated at the density of 3.5 × 104/ml in 96-well plates and incubated at 37°C in a 5% CO2 atmosphere for 24 h. Samples or recombinant TNF (as the standard) were incubated for a further 24 h, in the presence of 2 μg/ml actinomycin D. Cell survival was determined by measuring optical density at 630 nm after crystal violet staining. The diluted samples were assayed in triplicate. The percentage of cell destruction at a particular dilution was calculated as (Acont – Adil/Acont) × 100, where Acont is absorbance in control wells and Adil is absorbance in wells of a particular dilution of sample. The percent cytotoxicities were plotted against the logarithm of sample quantity. One unit of TNF activity was defined as the sample quantity required to achieve 50% cytotoxicity in the reaction.
Inhibition of Metastasis in Mice
The tumor-bearing C57BL/6 mice were sacrificed on the 21st day. Lungs were removed and fixed in 4% formalin. Tumor colonies were counted under microscope (× 200).
Platelet Aggregation Assay
Platelet aggregation was monitored by measuring electric impedance  using a whole-blood aggregometer (model OX-200; Shanghai Medical University Instrument Factory, Shanghai, China). On day 21, heparinized blood was drawn from tumor-bearing C57BL mice by cardiac puncture. Whole blood was then diluted with an equal amount of normal saline. The sample was placed in a plastic cuvette containing a magnetic stir bar and was kept at 37°C for 5 minutes before analysis. The platelet aggregation was then initiated by adding 10 μl 2 μmol/L ADP and monitored for up to 5 minutes.
Measurement of TXA2 and PGI2
On day 21, blood samples were collected from the femoral artery of the tumor-bearing C57BL/6 mice and transferred into a plastic tube prefilled with EDTA-Na2 (9:1). The tube was centrifuged at 1200 × g at 4°C for 10 min. The supernatant was frozen at -20°C. TXA2 with a half-life of approximately 30 sec under physiologic conditions was measured using its stable metabolite TXB2, and PGI2 using its stable metabolite 6-keto-prostaglandin F1α . All determinations were performed using commercially available 125I-TXB2 and 125I-Keto-PGF1α radioimmunoassays (RIA kits, Suzhou Medical College, Suzhou, China).
The data are expressed as mean ± S.D. Student's t-test or Dunnet t-test was used to compare the differences between treated groups and control groups, and differences were considered significant at P < 0.05.