Cell lines and cell culture
The cisplatin-resistant ovarian cancer cell line C13* and its parental variant OV2008 were provided by Doctor Benjamin K. Tsang (Department of Obstetrics, Gynecology and Cellular and Molecular Medicine, University of Ottawa, Canada). Cells were maintained in RPMI-1640 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere containing 5% CO2.
From 2004 through 2007, 40 patients who were diagnosed with ovarian carcinoma and received standard cisplatin-based intravenous chemotherapy after surgery at Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology were included in the study after obtainment of oral and written informed consents. Paraffin-embedded tumor tissue sections of each patient were prepared by the Department of Clinical Pathology. Information on histopathologic diagnosis was extracted from medical records and reviewed by a specialist in gynecologic pathology. The patients were selected and divided into treatment response and treatment non-response groups according to the CA125 criteria proposed by the Gynecological Cancer Intergroup (GCIC) . Briefly, treatment response patients were defined as having at least a 50% reduction in CA125 levels, when compared to pretreatment samples, and this reduction must have been maintained for at least 28 days; the intervening value of CA125 must have been less than or equal to the previous value. Patients demonstrated any clinical evidence of progression, such as increased lump size and ascites, were excluded from the treatment response group. The remaining patients were classified as treatment non-response group. This study was reviewed and approved by the ethics committee of the medical faculty at the Tongji Hospital, Tongji Medical College, Huazhong University of Science & Technology.
Antibodies and reagents
Antibodies against Akt, Ser473-phosphorylated Akt and p21 were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). β-actin antibody and secondary goat anti-rabbit and goat anti-mouse alkaline phosphatase antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-caspase 3 antibody was purchased from Biolegend Co. (San Diego, CA, USA). RPMI-1640, FBS, Lipofectamine 2000 and Trizol reagent were purchased from Invitrogen Co. (Carlsbad, CA, USA). Cisplatin, MTT, DMSO, G418 and DAPI dye were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The NE-PER cytoplasmic protein extraction kit was purchased from Pierce Co. (Rockford, IL, USA). The annexin-V/PI apoptosis detection kit was purchased from Promoter Biotech Ltd. (Wuhan, Hubei, China).
Construction of plasmids
A constitutively active Akt expression vector (AAkt2) and short hairpin RNA targeting Akt2 (Akt2Sh) were described previously [11, 35]. The sequences of the p21 RNA interfering fragment (p21si) were as follows: sense, 5'- CUU CGA CUU UGU CAC CGA G -3'; anti-sense, 5'- C UCG GUG ACA AAG UCG AAG -3' . The sequences of the mismatched fragment (p21sm) were: sense, 5'- CUC GAC UUC GUA CCC GAG -3': anti-sense, 5'- CUC GGG UAC GAA GUC GAG -3'.
Transient transfection for RNAi targeting
For RNAi targeting, C13* cells cultured in 6-well plates were transfected with indicated plasmids using Lipofectamine 2000. After 6 hours of incubation, the transfection solution was removed, and was replaced with fresh complete growth medium. 48 hours post-transfection the cells were assayed for the expression of p21 and treated with cisplatin for further experiment.
Establishment of stable-expression cell lines of AAkt2 in OV2008 cells and Akt2sh in C13* cells
OV2008 cancer cells were stably transfected with AAkt2 vector, C13* cancer cells were stably transfected with Akt2sh, using Lipofectamine 2000. Their corresponding empty vectors, i.e., pcDNA3.1 and pEGFPC1, were transfected as negative control. The cells were selected with G418. The concentration of G418 for selection and maintenance was 600 μg/μl. After three weeks the G418-resistant cell pools were established and seeded into 100 mm dishes for further propagation.
Total RNA was isolated from each group of cells using Trizol Regent, according to the manufacturer's instruction. Real-time PCR amplifications were carried out using DNase I (Promega)-treated total RNA. Reactions were performed in a Stratagene MX3000P system using the Real-time PCR Master Mix (TOYOBO, Japan). P21 primer sequences were as follows: sense, 5'-CCT CTT CGG CCC GGT GGA C-3': anti-sense, 5'-CCG TTT TCG ACC CTG AGA G-3'. GAPDH primer sequences were as follows: sense, 5'-ACG GAT TTG GTC GTA TTG GG-3'; anti-sense, 5'-TGA TTT TGG AGG GAT CTC GC-3'.
Western blot analysis
Total proteins were extracted by lysing cells in buffer containing 50 mM Tris pH 7.4, 150 mM NaCl, 0.5% NP-40, 50 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 25 mg/ml leupeptin and 25 mg/ml aprotinin. The lysates were cleared by centrifugation, and the supernatants were collected. Cytoplasmic proteins were extracted using the N-PER cytoplasmic protein extraction kit according to manufacturer's instructions. Equal amounts of protein lysate were used for western blot analyses. Specific signals were visualized with NBT/BCIP.
Cell viability assay using MTT
Cells were seeded into 96-well plates, treated with different concentrations of cisplatin for 24 hours and then assessed using 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide. Cell viability was determined by measuring the optical absorbance of cells at 570 nm wavelength and normalizing the values to the corresponding controls.
Analysis of apoptosis by flow cytometry
Cells were harvested, washed with PBS and stained with the annexin-V/PI apoptosis kit according to manufacturer's instructions. Analysis of apoptotic cells was performed using a FACScan flow cytometer, and the data were analyzed using cell fit software.
Cells were trypsinized and plated onto chamber slides for 12 hours. After fixation in acetone, the slides were blocked with BSA, incubated with p21 antibody and rhodamine-conjugated secondary antibody and counterstained with DAPI. Slides were then observed on a confocal laser-scanning microscope (Olympus IX81, Japan). P21 staining was categorized as negative, nuclear or cytoplasmic according to a standard described before . Briefly, negative was defined as undetectable cytoplasmic or nuclear staining. Nuclear p21 was defined as the fraction of tumor cells with positive nuclear staining greater than or equal to that of positive cytoplasmic staining. Cytoplasmic p21 was defined as the fraction of cytoplasmic staining greater than that of nuclear staining. Five randomized fields were counted in order to calculate the percentage of cells that stained with anti-p21 antibody.
Paraffin-embedded tissue sections were deparaffinized. After antigen retrieval, slides were incubated with 3% H2O2 to inhibit endogenous peroxidase. Slides were then blocked with 5% normal serum and incubated with anti-p21 antibody. For qualitative identification of specific antibody staining, the DAKO Envision+ system was used, according to manufacturer's instructions, as described below. After detection with chromogen diaminobenzidine, sections were counterstained with hematoxylin and mounted. For the negative control, all incubation steps were identical except that PBS was used instead of primary antibody. The immunoreactivity of p21 was categorized as negative, nuclear or cytoplasmic according to the standard decribed before .
Western blot images were captured and quantified using the ChemiImager 5500 system from the Alpha Innotech Corporation (San Leandro, CA, USA).
All experiments were repeated three times. The relationship between patients' clinical characteristics and results of p21 immunohistochemistry was assessed using the chi-squared (χ2) test. Results expressed as mean ± SD were analyzed using the Student t test. Differences were considered significant when p < 0.05. Data were analyzed using SPSS software version 13.0 (SPSS Inc., Chicago, IL).