Cell lines and materials
HCCLM3, a human HCC cell line with high metastatic potential that originated from MHCC97, was established by the Liver Cancer Institute of Fudan University (Shanghai, China) . Stable red fluorescent protein-expressing HCCLM3 (HCCLM3-R) cells by infection with lentivirus containing full-length cDNA of red fluorescent protein were also established by our institute . SMMC7721 was established by the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. The cells were maintained at 37°C with a 5% CO2 in DMEM supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin, 100 mg/ml streptomycin). Rapamycin and bortezomib were purchased from LC Lab (Woburn, MA). Both drugs were dissolved in DMSO, and the final concentration of DMSO in the cell culture studies was 0.1% or less. Most of the in vitro assays were performed use the following concentration: rapamycin (10 ng/ml) and Bortezomib (100 nM) or indicated otherwise. The concentrations of rapamycin and bortezomib were based on previous study [8, 24]. Chemical inhibitor of p53, pifithrin-α (PFT-α) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies for western blot such as anti-Akt, anti-p-Akt Ser473 and anti-GAPDH were purchased from Cell Signaling Technology (Danvers, MA). Other antibodies such as anti-PCNA, anti-CD31 were obtained from Abcam (Hong Kong, China). Cell Counting kit and colorimetric TUNEL system were purchased from Dojindo (Kumamoto, Japan) and Promega (Madison, WI), respectively.
Cell proliferation assay
To determine the effect of rapamycin and bortezomib on cell proliferation, we used a tetrazolium reagent, 2-(4-indophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphophenyl)-2 H-tetrazolium monosodium salt (CCK8, Cell Counting kit). In brief, 1 × 103 cells were seeded in 96-well culture plates. After an attachment period of 24 h, the cells were cultured in the presence of vehicle, rapamycin (10 ng/ml), bortezomib (100 nM), or a combination of both for 72 h. At the time of 24 h, 48 h and 72 h, the cells were incubated with CCK8 reagent for 1 h at 37°C. The staining intensity in the medium was measured by determining the absorbance at 450 nm.
Cell cycle analysis
HCCLM3 and SMMC7721 cells plated on 20-cm2 tissue culture flasks were collected at 24 h after the incubation with vehicle, rapamycin (10 ng/ml), bortezomib (100 nM), or the combination of rapamycin and bortezomib. Then, the cells were fixed in 70% cold ethanol for 1 h and resuspended in a hypotonic propidium iodide (PI) solution (Sigma, St Louis, MO) containing RNase. Flow cytometry was performed with the use of Coulter epic flow cytometer. DNA histograms were analyzed using Modfit computer program (Verity Software House, Topsham, ME, USA). In addition, the percentage of apoptotic cells in the sub-G1 fraction was also calculated.
Analysis of apoptotic cells by Hoechst 33342 staining
The chromatin dye Hoechst 33342 was used to assess apoptotic cells. The cells were incubated with vehicle, rapamycin (10 ng/ml), bortezomib (100 nM), or the combination of rapamycin and bortezomib for 24 or 48 h. After washing three times with PBS, the cells were fixed in 4% formaldehyde at room temperature for 30 min, then washed twice with PBS and exposed to 5 μg/ml Hoechst 33342 for 30 min at room temperature. Afterwards, the cells were observed with a fluorescence microscope. Apoptotic cells were characterized by morphological alterations such as condensed nuclei and cell shrinkage.
Cell migration assay and cell invasion assay
Cell migration was performed by transwell assay (Corning Costar, Cambridge, MA). The cells were incubated with vehicle, rapamycin (10 ng/ml), bortezomib (100 nM), or the combination of rapamycin and bortezomib for 24 h. Then 5 × 104 pretreated 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 in the upper chamber were removed by cotton swab and the cells that had reached the underside of the membrane were fixed with 4% paraformaldehyde and stained with 10% Giemas for 10 min. The cells that located on the underside of the filter (5 fields/filter) were counted. The cell invasion assay was carried out similarly, except that the matrigel (BD Biosciences) was added to each well 6 h before cells were seeded on the membrane.
Western blot analysis
Equal amounts of total proteins (20 μg) were separated by 10% SDS-PAGE and transferred to a 0.45-mm PVDF membrane using a Bio-Rad SemiDry apparatus. The membranes were blocked at room temperature for 1 h with 5% nonfat milk in TBS containing Tween 20 (TBST). Then membranes were incubated with rabbit anti-Akt, rabbit anti-p-Akt Ser473, or mouse anti-GAPDH overnight at 4°C, followed by HRP-conjugated secondary antibodies for 1 h at room temperature. After washing three times in TBST, the membrane was incubated with Chemiluminescent Detection Reagent for 5 min and exposed to X-ray film.
Total RNA was extracted using Trizol Reagent (Invitrogen, Carlsbad, CA) and was reverse-transcribed into complementary DNA with oligo (dT)18 primer using TransScript first-strand cDNA synthesis kit (Transgene Biotech, China) according to the manufacturer’s instructions. PCR was performed with PCR kit (Lifefeng, China) according to the manufacturer’s instructions. The primer sequences, annealing temperature, number of cycles used for PCR, and length of the amplified products can be seen in Additional file 1: Table S1. The PCR products were analyzed on 1.5% agarose gel and visualized under UV light following ethidium bromide staining. Quantitative data were expressed by normalizing the densitometric units to GAPDH (internal control).
Orthotopic tumor model
Male BALB/C nude mice (5- to 6-week-old) were obtained from Shanghai Institute of Materia Medica (Chinese Academy of Sciences, Shanghai, China). The in vivo experiments were carried out strictly in accordance with a protocol approved by the Shanghai Medical Experimental Animal Care Committee (Permit Number: 2009–0082). HCCLM3-R hepatocellular cancer orthotopic xenograft model was established as described previously [29, 30]. Briefly, HCCLM3-R cells (5 × 106) were injected subcutaneously into the upper left flank region of nude mice. When the tumor reached 1 cm in diameter, they were cut into 2 × 2 × 2 mm3 sized pieces, and implanted into livers of 24 nude mice. The 24 mice were randomly assigned to four experimental groups (6 mice/group): control, rapamycin, bortezomib, and the combination of rapamycin with bortezomib. Both drugs were dissolved in DMSO and then diluted in saline, and the final concentration of DMSO in the in vivo studies was less than 0.1%. On day 4 after the tumor implantation, rapamycin was delivered orally at a dose of 2 mg/kg every day. Bortezomib was administered as i.p. injections at a dose of 0.5 mg/kg in 100 μL of 0.9% saline solution twice a week. The dosage of rapamycin and bortezomib is based on previous studies [9, 25]. The mice treated with both rapamycin and bortezomib was administered by using the same schedule as described for the single drug treatment. Control mice were received 100 μL of saline orally (every day) and 100 μL of saline by i.p. injections (twice a week) including the same concentration of DMSO with the treatment groups.
The treatment was stopped on day 33. Then the mice were sacrificed on day 35 after tumor implantation. At necropsy, tumor was measured for largest (a) and smallest (b) diameters, and the tumor volume was calculated as V = a × b2/2. The lungs were excised, and red fluorescent protein-positive metastatic foci were imaged by fluorescent imaging (Leica MZ6). Then, orthotopic tumors and lungs were fixed with 10% buffered formalin and embedded with paraffin. Lung metastasis was also determined by examining serial sections of every lung tissue block by microscopy.
Immunohistochemistrical staining for Akt, p-Akt, PCNA and CD31
Tumor tissue was fixed, embedded, and sliced into 5 μm thick sections. Immunostaining of Akt, p-Akt Ser473, PCNA and CD31 was carried out as described previously . Briefly, paraffin sections were first deparaffinized and then hydrated. After microwave antigen retrieval, endogenous peroxidase activity was blocked with incubation of the slides in 0.3% H2O2, and non-specific binding sites were blocked with 10% goat serum. Then the sections were incubated with primary antibodies (rabbit anti-Akt, rabbit anti-p-Akt Ser473, rabbit anti-PCNA and mouse anti-CD31) overnight at 4°C, followed by HRP-conjugated secondary antibodies for 1 h at room temperature. Finally, the sections were developed in diaminobenzidine solution under a microscope and counterstained with hematoxylin. The proliferation index was determined as number of PCNA-positive cells/total number of cells in 5 randomly selected high-power fields (magnification × 200).
In situ apoptosis detection by TUNEL staining
Paraffin-embedded, 5 μm thick sections were used to identify apoptotic cells by staining using TUNEL assay kit according to the manufacturer’s instructions. The extent of apoptosis was evaluated by counting the TUNEL-positive cells (brown-stained). The apoptotic index was determined as number of TUNEL-positive cells/total number of cells in 5 randomly selected high-power fields (magnification × 200).
Analysis was performed with SPSS 15.0 for Windows. In vitro cell proliferation, apoptosis migration and invasion assays were analyzed by Student's t-test. Tumor volumes and histologic quantitations were also compared by Student’s t test. Categorical variables were tested with the use of Fisher’s exact test. P <0.05 were considered statistically significant.