Cell lines
In the present study, we used two human HCC cell lines: MHCC97L cells, which originated from MHCC97 [12, 13] (established at the Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China), and HepG2 cells (American Type Culture Collection). MHCC97L cells show a moderate metastatic potential, and HepG2 cells show a low metastatic potential. All cells were cultured with Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and incubated in a humidified incubator at 37°C in 5% CO2.
Compounds and antibodies
Oxaliplatin was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Monoclonal antibodies used in immunofluorescence, immunoblotting, and immunohistochemistry included: rabbit anti-human vimentin, Twist, and Slug (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-human E-cadherin and Snail (Abcam Ltd., Cambridge, UK), mouse anti-human N-cadherin (Abcam Ltd, Cambridge, UK.), mouse anti-human GAPDH (Chemicon, CA, USA) and mouse anti-human Ki-67 (Dako, Glostrup, Denmark).
Treatment of tumor cells with oxaliplatin and analysis of cell morphology
MHCC97L or HepG2 cells were plated in six-well tissue culture plates (1 × 105 cells/well). After 24 h, the medium was replaced with DMEM containing 10% FBS and 2 μmol/L oxaliplatin. After 48 h, the medium was changed, and drug treatment was terminated. Cells were allowed to recover, and when the surviving populations reached 80% confluence, cells were passaged and exposed to oxaliplatin again for 48 h. During 6 weeks of treatment this process was repeated for a total of four 48-h exposures to oxaliplatin. Cells surviving treatment (Oxa cells) were designated MHCC97L-Oxa and HepG2-Oxa, respectively. The morphological characteristics of parental and Oxa cells were compared by microscopy (Olympus, Tokyo, Japan).
Cell migration and invasion assays
Cell migration was assessed by transwell assay (Boyden chambers) (Corning, Cambridge, MA, UK). Briefly, 6 × 104 cells in serum-free DMEM were seeded on a membrane (8.0-μm pore size) inserted in a well of a 24-well plate. DMEM containing 10% FBS was added to the lower chamber of each well. After 48 h, cells that had reached the underside of the membrane were stained with Giemsa (Sigma Chemical Co.) and counted at × 200 magnification. The cell invasion assay was carried out similarly, except that 10 μL of matrigel (BD Biosciences) was added to each well 6 h before cells were seeded on the membrane.
Cell proliferation assay
MHCC97L and HepG2 parental and Oxa cells were incubated in 96-well plates (5 × 103 cells/well) for 24, 48, or 72 h. Cell proliferation was then determined with an MTT kit (Beyotime, Shanghai, China). Results were expressed as the absorbance of each well at 570 nm (OD570).
Western blot analysis and immunofluorescence
The concentration of protein extracted from Oxa and parental cells was determined with the BCA Protein Assay Kit (Beyotime, Shanghai, China). The expression of E-cadherin, N-cadherin, vimentin, Snail, Slug, and Twist was determined by immunoblotting as previously described [14].
E-cadherin, N-cadherin, and vimentin expression in parental and Oxa cells were also demonstrated by immunofluorescence. Cells were grown on glass cover slips to 40%-50% confluence, and then fixed, permeabilized, and blocked. Cells were then incubated with primary monoclonal antibodies against E-cadherin, N-cadherin, and vimentin overnight at 4°C. The next day, slides were washed and incubated with anti-mouse and/or anti-rabbit fluorescein isothiocyanate (FITC)- and/or tetramethyl rhodamine isothiocyanate (TRITC)-conjugated secondary antibody (Invitrogen). Cells were counterstained with 4'-6-diamidino-2-phenylindole (DAPI) to visualize cell nuclei and visualized by fluorescence microscopy (Olympus, Tokyo, Japan).
Animals
Male BALB ⁄ c nu ⁄ nu mice (age, 4-6 weeks old; weight, approximately 20 g) were obtained from the Shanghai Institute of Materia Medica (Chinese Academy of Science) and maintained under standard pathogen-free conditions. The experimental protocol was approved by the Shanghai Medical Experimental Animal Care Commission.
Pilot study for animal model
To evaluate changes in metastatic potential of residual HCC cells after in vivo oxaliplatin treatment, residual cancer from the livers of oxaliplatin-treated and untreated nude mice were re-inoculated orthotopically into new recipient nude mice. Because the treated and untreated tumors needed to be histologically similar, a pilot study compared the histology of treated and untreated tumors at different time points after chemotherapy to determine the appropriate time points for re-inoculation.
A metastatic model of human HCC in nude mice using MHCC97L cells was employed for this pilot study [12]. Briefly, MHCC97L cells (5 × 106) were injected subcutaneously into the upper left flank region of nude mice. When the subcutaneous tumor reached approximately 1 cm in length (approximately 4 weeks after injection), it was removed, minced into small pieces of equal volume (2 × 2 × 2 mm3), and transplanted into the livers of 60 different nude mice. Based on the literature [15] and the results of our previous studies, a dosage for oxaliplatin of 10 mg/kg, once a week was adopted in the present study. Oxaliplatin was administered intraperitoneally (i.p.) to randomly selected mice (oxaliplatin treatment group; n = 30) on days 12, 19, and 26 after inoculation; the control group (n = 30) received 0.2 mL of 0.9% sodium chloride (i.p.) on those days. On days 1, 2, 5, 7 and 14 after the final treatment, six mice from each group were sacrificed by cervical dislocation. The necrosis and apoptosis of tumors in each group were compared.
Analysis of tumor necrosis and apoptosis
Paraffin-embedded sections were prepared for hematoxylin and eosin staining. Necrosis of tumor tissue was determined by comparing the surface of necrotic areas to that of the whole tumor [16]. Apoptosis was determined using a terminal transferase dUTP nick end labeling (TUNEL) assay kit (KeyGen, Nanjing, China) according to the manufacturer's protocol. The apoptosis rate was expressed as a ratio of apoptotic cells to total tumor cells.
Re-inoculation experiment
Analysis of tumor histology in the pilot study showed that tumor necrosis and apoptosis on days 5, 7 and 14 after the final oxaliplatin treatment were not significantly different between the control group and the treated group. We therefore used day 7 tumors for re-inoculation. Orthotopic models of human HCC were established in 36 nude mice and treated with oxaliplatin (n = 18) or sodium chloride (n = 18) using the same protocol described for the pilot study. On day 7 after the final treatment, all mice were sacrificed by cervical dislocation. Tumor fragments of equal volume (2 × 2 × 2 mm3) from each mouse of the oxaliplatin-treated and untreated control groups were re-inoculated into the livers of each new recipient mice correspondently. The remaining tumor tissues of both groups were fixed in 10% buffered formalin and embedded in paraffin wax for histological study.
These mice, segregated into control (bearing untreated tumors, n = 18) and trial ( bearing oxaliplatin pre-treated tumors, n = 18) groups, were then kept under standard conditions. On day 42 after re-inoculation, 12 randomly selected mice from each group were sacrificed, and tumor growth and pulmonary metastasis were assessed. Tumor tissues from re-inoculated mice were also prepared for histological study. The remaining six mice from each group were kept for survival analysis.
Characterization of the Chinese herbal extract Songyou Yin
The water-soluble Chinese herbal medicine Songyou Yin (SYY), authorized by the Chinese State Food and Drug Administration (Grant No. G20070160), includes five Chinese medicinal herbal extracts in the following proportions (w/w): Salvia miltiorrhiza Bge., 14.3%; Astragalus membranaceus Bge., 14.3%; Lycium barbarum L., 23.8%; Crataegus pinnatifida Bge., 23.8% and Trionyx sinensis Wiegmann, 23.8% (all from China). High-performance liquid chromatography (HPLC) fingerprinting of Songyou Yin and its five characteristic components (see additional file 1) was carried out by the Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences (CAS), China [17]. The SYY used in this study was produced by the Caitong Detang Chinese Traditional Medicine Pharmaceutical Factory (batch number: 090401; Shanghai, China).
Songyou Yin treatment in the re-inoculation model beaing oxaliplatin pre-treated tumors
The orthotopic nude mice model bearing oxaliplatin pre-treated MHCC97L xenografts was established in 84 mice using the previously described protocol. Three days after re-inoculation, mice were randomized into control (n = 21) and SYY-treated groups (n = 63). The 63 nude mice in the SYY-treated group received SYY at three different doses (2.1, 4.2, and 8.4 g/kg; each dose, n = 21) by oral gavage once per day. Mice in the control group received 0.2 mL of 0.9% sodium chloride via oral gavage at the same time. Treatment continued for 6 consecutive weeks. On day 42 after initiation of treatment, mice from the control group and each SYY dose group (each group, n = 15) were sacrificed to determine tumor volume. The tumor tissue and lungs were collected for histological analysis and a pulmonary metastasis assay. The remaining mice were maintained and used for survival analysis.
Tumor volume, lung metastasis and survival
Tumor volume was calculated as: (a × b2)/2, where "a" is the widest diameter and "b" is the smallest [18]. Lung metastasis was determined by examining serial sections of every lung tissue block by microscopy. Survival time was defined as the interval between the day of inoculation and the day of death.
Immunohistochemistry
Tumor tissue was fixed, embedded, and sliced into 5-μm thick sections. Immunostaining of E-cadherin, N-cadherin, vimentin, Snail, Slug, and Twist was carried out using a standard protocol [19].
Statistical analysis
In vitro cell migration, invasion, and proliferation assays were analyzed by Student's t-test. Necrosis and apoptosis of tumors from the animal model were also compared by Student's t-test. Tumor volume was compared by analysis of variance (ANOVA), the lung metastasis assay was analyzed using Fisher's exact test, and survival was compared with Kaplan-Meier method with a log-rank test. Statistical analysis was performed with SPSS 15.0 for Windows (SPSS Inc. Chicago, IL, USA). P < 0.05 was considered statistically significant.
