Cell lines, media and plasmids
HOC-7 with an average doubling time of 16.4 days is a kind gift from Dr R. N. Buick (University of Arizona, Tucson), which was derived from ascites of a patient with a well-differentiated Stage III serous adenocarcinoma of the ovary [9, 14]. SKOV3 human ovarian cancer cell lines were bought from the American Type Culture Collection (Rockville, MD). HOC-7 and SKOV3 cell lines were cultured in Minimum Essential Alpha 1×/ RPMI-1640 medium (GIBCO BRL, Life Technologies, Gaithersburg, MD) supplemented with 10 % fetal bovine serum (HyClone, Logan, UT) at 37 °C with 5 % CO2. Both HOC-7 and SKOV3 were transfected with dominant negative mutant IĸB, henceforth IĸBαM, using N-[1-(2,3-dioleoyloxyl)propyl]-N,N,N-trimethylammoniummethyl sulfate liposomal transfection reagent (Boehringer Mannheim, Indianapolis, IN) and selected by neomycin antibiotics (2.5ug/ml) for 14 days. The plasmid used for stable transfection was a pBabe-U6/puromycin vector (5.4 kb) containing IkBαM under cytomegalovirus (CMV) promoter expression control. The primers used to sequence IkBαM were p1: catatggatccatgtttcagccagctgggcacg p2: cctatctcgagttataatgtcagacgctggcctc. The mutation place in the serines 32 and 36 of IkBα were replaced by glycine and alanine, which damaged the phosphorylation and followed proteolysis, eventually led to the irreversible binding to Rel/NF-kB and inhibited its translocation in nuclei [9, 15–18]. In addition, we transfected HOC-7 and IkBαM expressing HOC-7 cell line with p53 siRNA, the positive colony were selected by puromycin (300ug/ml). SiRNAs against p65 and p53 and control siRNA were purchased from Santa Cruz Biotechonology.
Mutantional analysis of KRAS in HOC-7 and SKOV3 cell lines
Genomic DNA was purified from all of the HOC-7 and SKOV3 cell lines using the QIAGEN’s DNeasy Tissue Kit (QIAGEN Inc, Chatsworth, CA, USA). PCR was performed ensued by nucleotide sequencing using the iCycler (Bio-Rad, Hercules, CA, USA), which is including the exon 1 of KRAS. The primers used to sequence were: 5-taaggcctgctgaaaatgactg-3 (forward); 5-tggtcctgcaccagtaatatgc-3 (reverse); and 5-ctgcaccagtaatatgcatattaaaac-3 (sequencing); The thermal profile was: first denaturation step 95 °C 2 min, followed by 95 °C 30 s, 58 °C for 30s, 72 °C for 35 s, total 35 cycles; 72 °C for 10 min [19, 20]. The Lasergene program (DNASTAR, Madison, WI) was used to analyze these sequences.
Electrophoretic mobility shift assay (EMSA) and luciferase reporter assay
The electrophoretic mobility shift assay was performed as described before. Briefly, the nuclear extracts of different cell lines were prepared by lysed in nuclear extract buffer (20 mM HEPES, 400 mM NaCl, 1 Mm EDTA, 1 Mm EGTA, 10 μM dithiothreitol, 20 μg/ml leupeptin, 20 μg/ml aprotinin, 500 μg/ml benzamidine) and the concentrations were determined using the Coomassie brilliant blue G250 assay kit. Double-stranded deoxyoligonucleotides containing the NF-kB consensus recognition site (5′-AGT TGA GGG GAC TTT CCC AGG C-3′ and 5′-GCC TGG GAA AGT CCC CTC AAC T-3′, Santa Cruz Biotechnology Inc, Santa Cruz, CA) were labeled with (r-32p)ATP using T4 polynucleotide kinase according to the protocol. The nuclear probe was incubated with the radiolabeled probe DNA (3.5pmol, 10 μCi) and 4 μl 5 × binding buffer. For the supershift, the proteins were incubated with p65 or p50 monoclonal antibody before 32p labeled. All of the DNA-protein complexes were resolved on 5 % PAGE in Tris/glycine buffer after incubated at room temperature for 30 min. For the NF-kB transcription assay, seeded cells were precultured to 75 % confluence and transfected with and HIV promoter-driven luciferase cDNA plasmid, and then tested according to promega kit.
Cell proliferation assay
Cell proliferation was determined by examined by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide assay (MTTassay; Sigma) with the CellTiler 96 Aqucous One Solution Cell Proliferation Assay Kit (Promega) according to the manufacturer’s protocol. Four different cell lines HOC-7, HOC-7 IĸBαM, HOC-7 P53i and HOC-7 IĸBαM P53i were seeded at 5 × 103 cells/well in 96-well tissue culture plates. Cell growth curve was assessed every 24 h and totally 3 days.
Soft agar colony formation assay
Soft agar growth assay was performed as previously described (Difco, Detroit, MI). The four cell lines were mixed with 0.4 % agar were overlaid above the supporting 0.6 % agar and cultured total 25 days with replenished medium. Random fields were recorded using a Leica TCS4D confocal scanning laser microscope (Leica, Solms, Germany) and the total number and the size of the colonies were counted.
Western blot analysis
Western blot analysis was performed as preciously described. Lysates of treated cells or tumor samples were collected and lysed in radioimmunoprecipitation assay buffer. Equal amounts of proteins were resolved on SDS-PAGE and then transferred onto a polyvinylidene difluoride membrane. Specific primary antibodies were used for the following molecules : IĸBα(Cabiochem, San Diego, CA, USA), p65, Iĸĸα, Iĸĸβ, AKT, p-AKT, MEK, p-MEK, ERK, p-ERK, p38, caspase-10, caspase-1, cyclinA, cyclinB, PUMA, BID, CIAP, Bad, p-Bad, PKC (Cell signaling Technology), JNK2/1, p-JNK, TSP1, Bcl-2, Bcl-xL, Bax, APAF1, Fas, caspase-8, Mrg1, cyclinD, p53, MMP1 (Santa Cruze Biotechnology, CA), caspase-9, caspase-3 (BioLegend). β-Actin (Santa Cruze Biotechnology, CA) was used to assess the equal loading. The phosphorylated protein controls obtained from blots that had been stripped and re-probed for the same sample lane. Sixty microgram of cell protein extract was loaded per lane. CS-710 Calibrated Imaging Densitometer (Bio-Rad) was utilized for densitometric quantification.
Apoptosis detection
Derivative cells and control cells were cultured to 80 % confluence. Cells were harvested after being washed three times with ice-cold phosphate-buffered saline (PBS). Subsequently, cells were labeled with Annexin V and propidium iodide according to the manufacturer’s protocol (BD ApoAlert Annexin V-FITC Apoptosis kit; BD Biosciences, PaloAlto, CA) [21]. The percentage of apoptotic cells was determined by (M2) peak in the histogram generated by FACSCalibur system and Cell Quest software (BD Biosciences).
Tumor xenograft in nude mice
The xenograft tumor model has been described earlier [22]. Briefly, 5 × 106 cells of either wild type HOC-7, SKOV3, IĸBαM expressing HOC-7 and SKOV3 or P53-inactivating cells were subcutaneously injected into 4- to 6-week-old female BALB/athymic nude mice (NCI-Frederick, NIH). Each group includes 8 mice. One week after implantation, the mice were checked every 2 days per week. The control group received saline (PBS) only. The same quantity of saline was used in these groups to resuspend different cell lines. General clinical observation of the mice included determination of syndrome, side effect and body weight. To determine tumor size, we measured two perpendicular diameters of the xenograft in centimeters by calibers. Tumor weight was then estimated using formula 1/2a × b2, where a is the long diameter and b is the short diameter [23, 24]. 66 days after the first injection, all mice were euthanized by carbon dioxide asphyxiation and the tumor tissues were processed for immunohistochemistry staining followed by 10 % formalin fixation overnight. All protocols for animal use were reviewed and approved by the Animal Care Committee of West China Second University hosipital in accordance with Institutional Animal Care and Use Committee guidelines.
Patient tissue specimens
The use of tissue blocks and chart reviews were approved by the Institutional Review Board of Sichuan University. The specimens, including controls, used in our study were preserved and stored by the Tissue Bank Core Facility at Sichuan University. Detailed quality control procedures for tissue sampling were chosen and implemented by the Tissue Bank Core Facility at Sichuan University. Patients had been treated with either chemotherapy or irradiation by the treating physicians, and the selection of patient tissues was not based on the treatments. Follow up information was updated through March 2011 by reviewing medical records. The randomly selected formalin-fixed paraffin-embedded tissues included normal ovarian tissues (n = 20) and low-grade serous ovarian carcinomas (n = 416) were not matched. Tumor sample collection and tissue microarray construction have been described previously [25, 26]. Briefly, ovarian tissue microarray blocks were diagnosed in duplicate by two gynecologic pathologists who reviewed hematoxylin and eosin–stained sections and constructed by taking core samples from morphologically representative areas of paraffin-embedded tumor tissues and assembling them on a recipient paraffin block. The follow-up protocol have been mentioned previously, the same as the characteristics of exclusion criteria and eligible participants. All of the participants provided written informed consent and the study protocol was agreed by the institutional review boards of Sichuan University (no.11789).
Immunohistochemical staining and analysis
The streptavidin-peroxidase immunohistostaining method for NF-kB p65 was performed as described elsewhere [27, 28]. Briefly, samples were fixed in 10 % formalin buffer and embedded in paraffin. Tissue sections (4 μm thick) were steamed in universal decloaker (Biocare Medical, Walnut Creek, CA, USA) for antigen retrieval, followed by 19 min protein-blocking (Biocare Medical). All slides were first incubated against NF-kB p65 (1:500, for 1 h at room temperature; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and then treated with secondary antibody (Biocare Medical) and horseradish peroxidase for 15 min each. The tissues were stained for 3 min with high sensitivity 3,3′-diaminobenzidine tetrahydrochloride, counterstained with hematoxylin, dehydrated and then mounted [29, 30].
Statistical analysis
Statistical analysis was performed by using Fisher’s exact test at different time points for the mean tumor sizes of each group. X-tile software was used to find the optimal cut-off point. Monte Carlo simulations was used to determine the prognostic significance. Disease-specific survival rates were calculated by the Kaplan-Meier method and compared by the log-rank test. Cox proportional hazards regression models in Statistica software (SAS Institute, SAS Language Reference, version 8, SAS Institute, Inc) were used for multivariate analyses of survival. The rest of statistical analysis was performed by using Student’s t-test (STATISTICA6 software, StatSoft, Tulsa, OK). Differences with P-value was less than 0.05 were considered significant. All statistical tests were two-sided.