Overexpression of FADD enhances 5-fluorouracil-induced apoptosis in colorectal adenocarcinoma cells
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- Yin, A., Jiang, Y., Zhang, X. et al. Med Oncol (2010) 27: 397. doi:10.1007/s12032-009-9224-x
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To investigate the mechanism of enhancing apoptosis-inducing effects of 5-fluorouracil on human colorectal adenocarcinoma cells by stable transfection of extrinsic Fas-associated death domain protein (FADD) gene, both in vitro and in vivo. FADD gene of stable overexpression was determined by reverse transcription polymerase chain reaction (RT-PCR) assay and Western blotting assay. After treatment with 5-fluorouracil as an apoptotic inducer, in vitro cell growth activities were investigated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Cell apoptosis and its rates were evaluated by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay and flow cytometry of annexin V-FITC/PI staining. To examine the combination therapeutic effect of FADD and 5-fluorouracil, tumor xenograft model was prepared for in vivo study. Compared with SW480 and SW480/neo cells, FADD mRNA and protein levels of SW480/FADD cells were higher. Chemosensitivity and apoptosis rates of SW480/FADD cells were remarkably higher than SW480 and SW480/neo cells when treated with 5-fluorouracil. In in vivo study, overexpression of FADD increased the efficacy of 5-fluorouracil-induced inhibition of tumor growth in nude mice. Stable overexpression of extrinsic FADD gene can conspicuously ameliorate apoptosis-inducing effects of 5-fluorouracil on colorectal adenocarcinoma cells, which is a novel strategy to improve chemotherapeutic effects on colorectal cancer.
KeywordsFas-associated death domain protein (FADD)5-FluorouracilApoptosisColorectal adenocarcinoma
Colorectal cancer is the second leading cause of cancer-related mortality in the United States . In 2008, an estimated 148,810 individuals have been diagnosed with the disease and 49,960 have died of causes related to colorectal cancer . Despite advances in medical practices, survival rates of colorectal cancer have changed little in the last 20 years . Therefore, there is an acute need to explore novel forms of the treatment for colorectal cancer.
Up to now, chemotherapy is still the important adjuvant treatment for postoperative and advanced colorectal cancer. In the treatment of colorectal cancer, 5-fluorouracil, a potent inhibitor of thymidylate synthesis during DNA synthesis , is one of the most commonly used chemotherapeutic agents. More findings have confirmed the clinical efficacy and cost-effectiveness of a 6-month regimen of 5-fluorouracil-based chemotherapy in improving rates of disease recurrence (by 34%) and overall survival (by 24%) . However, the clinical efficacy of 5-fluorouracil is often limited by the rapid development of resistance, and the factors that determine the risk of developing resistance are largely unknown. Therefore, drug resistance is a severe obstacle for the success of using 5-fluorouracil to treat colorectal cancer.
Fas-associated death domain protein (FADD), also named Mort1, which was discovered by Chinnaiyan et al.  and Boldin et al. , plays an essential role as an adapter molecule in Fas (CD95/APO-1)-mediated apoptosis and contributes to anticancer drug-induced cytotoxicity. FADD comprises two domains: death effect domain (DED) and death domain (DD) , which recruits initiator caspase-8, by interaction via DED, present in the prodomain of caspases to form the death-inducing signal complex (DISC) . Overexpression of FADD has been shown to induce apoptosis in malignant glioma cells  and rheumatoid synoviocytes .
In this study, we addressed the hypothesis that FADD plays a synergistic role in 5-fluorouracil-induced apoptosis in colorectal cancer cells. To test our hypothesis, we constructed a SW480/FADD cell line using G418 selection in human colorectal cancer SW480 cells. Cellular responsiveness to 5-fluorouracil was examined using various assays in both non-transfected SW480 cells and the FADD-transfected SW480 cells. And in vivo study, colorectal tumor xenograft model was constructed to investigate the combination therapeutic effect of FADD and 5-fluorouracil.
Materials and methods
Cell and cell culture
Human colorectal carcinoma cell lines, SW480, were purchased from Shanghai Cell Collection (Shanghai, China). Cells were grown in RPMI1640 medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco BRL, Grand Island, NY, USA), in a humidified atmosphere at 37°C and 5% CO2.
Vector construction and transfection
FADD full-length cDNA was amplified by RT-PCR with total RNA extracted from human colorectal carcinoma SW480 cells as template, the primers that included forward (5′-TTT GGA TCC GCT AGC ATG GAC CCG TTC CTG GTG CTG-3′) and reverse (5′-TTT CTC GAG TCA GGA CGC TTC GGA GGT AGA-3′) sequences (94°C for 30 s, 56.5°C for 45 s, 72°C for 30 s). Full-length FADD cDNA (651 bp) was then cloned into the HindIII/EcoRI sites of the eukaryotic expression vector pEGFP-N1 (4.7 kb) (Clontech, San Jose, CA, USA). SW480 cells were transfected by LipofectamineTM 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Cells were then grown in complete medium containing 250 mg/ml G418 (Sigma, St.Louis, MO, USA). Colonies were isolated and expanded into cell clones at the end of 4 weeks. The subclone cells expressing FADD and neo genes were named as SW480/FADD cells and SW480/neo cells, respectively.
mRNA expression levels were analyzed by RT-PCR assay. Total RNA (1 μg) was isolated by Trizol reagent kit (Invitrogen, Carlsbad, CA, USA) and reverse transcription was performed with the First Strand cDNA Synthesis Kit (TOYOBO, Osaka, Japan) according to the manufacturer’s instructions. PCR was performed with primers for FADD (F: CCG CCA TCC TTC ACC AGA; R: CAA TCA CTC ATC AGC ACC TCA; 304 bp; GenBank NM_003824.2), GAPDH (F: GGA TTT GGT CGT ATT GGG; R: GGA AGA TGG TGA TGG GAT T; 205 bp; GenBank NM_002046.3). GAPDH was used as an internal control. Initial denaturation was done at 94°C for 5 min followed by 35 cycles of amplification. Amplification protocol was repeated cycles of denaturation (30 s, 94°C), annealing (45 s; 56.5°C for FADD and 51.4°C for GAPDH), extension (30 s, 72°C) and final extension (7 min, 72°C). PCR products were electrophoresed through 1.5% agarose gels containing ethidium bromide (0.5 μg/ml). Gels were visualized under UV light, photographed and optical densities of the bands were analyzed using the Quantity One software (Bio-Rad, Hercules, CA, USA).
Western blotting assay
Protein expression levels were analyzed by Western blotting assay. Briefly, cells were washed with PBS and lysed in lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM EDTA, 50 μg/ml leupeptin, 30 μg/ml aprotinin, and 1 mM PMSF). A total volume of 40 μg of protein was loaded per lane and separated on 12.5% sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gels and then transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA) by wet transfer system (Bio-Rad, Hercules, CA, USA). After the membrane was blocked with 10% nonfat dry milk in TBST and then incubated with primary antibodies: procaspase-8, cleaved caspase-8, procaspase-3, cleaved caspase-3, and actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. The following dilutions were used: procaspase-8 1:1000, procaspase-3 1:1000, cleaved caspase-8 1:500, cleaved caspase-3 1:500, and actin 1:3000. After three washes with TBST, the membranes were incubated with biotinylated goat anti-rabbit IgG secondary antibody (Promega, Madison, WI, USA, 1:5000 dilution) for 1 h at room temperature and then developed with the use of an enhanced chemiluminescence (ECL) system (Millipore, Bedford, MA, USA) and then exposed with Kodak X-ray film. Protein band intensities were determined densitometrically using the video imaging CMIASWIN system (Bio-Rad, Hercules, CA, USA).
Cell viability assay
SW480, SW480/neo, and SW480/FADD cells were seeded at 6 × 105/l density into 96-well chamber slides. For each cell line, 0.1 mg/l 5-fluorouracil (Sigma, St.Louis, MO, USA) group, 1 mg/l 5-fluorouracil group and 10 mg/l 5-fluorouracil group were designed, with each group having five wells. After treating with 5-fluorouracil for 24 h, 20 μl (5 g/l) MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Sigma, St.Louis, MO, USA) was added into each well and cultured for another 6 h, the supernatant was discarded, then 100 μl DMSO (Sigma, St.Louis, MO, USA) was added. When the crystals were dissolved, the optical density A values of the slides were read on the enzyme-labeled minireader II (Bio-Rad, Hercules, CA, USA) at the wave length of 490 nm. Cellular growth inhibitory rate (%) = (1−average A490 nm value of experimental group/average A490 nm value of control group) × 100%. For each detection, the total procedure was repeated 3 times.
Cell apoptosis analysis
After treatment with 10 mg/l 5-fluorouracil for 48 h, apoptosis of SW480, SW480/neo, and SW480/FADD cells were evaluated by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay, using an in situ cell death detection kit (Roche Diagnostics, Branchburg, NJ, USA). Briefly, cells were grown on 4-well chambered slides, washed twice with ice-cold PBS, and fixed with 4% paraformaldehyde in PBS for 1 h at room temperature. After washing twice with PBS, the cells were treated with permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 10 min on ice. Samples were then washed twice with PBS and incubated with the TUNEL reaction mixture, containing FITC-labeled dUTP and terminal deoxynucleotidyl transferase, for 1 h at 37°C in a humidified atmosphere in the dark. Control cells were incubated in the absence (negative control) or presence (positive control) of DNAse, in addition to control samples incubated without deoxynucleotidyl transferase. After incubation, the cells were washed twice with PBS and analysed using the fluorescence microscopy system (Carl Zeiss, Thornwood, NY, USA). Use an excitation wavelength in the range of 488 nm and detection in the range of 550 nm. Three random fields were counted per well in duplicate wells from three experiments.
After treatment with 10 mg/l 5-fluorouracil for 48 h, apoptotic ratios of SW480, SW480/neo, and SW480/FADD cells were determinated by annexin V-FITC and propidium iodide (PI) (BioVision, Mountain New, CA, USA) staining flow cytometry. Cells from the above groups were collected, washed twice with cold PBS, resuspended with 100 μl binding buffer into 5 × 105/ml, and incubated with annexin V-FITC at room temperature for 15 min. After washing with binding buffer, the cells were resuspended with 500 μl binding buffer containing 10 μl PI (20 μg/ml), and incubated on ice for 30 min. Apoptosis was analyzed by flow cytometry at a wavelength of 488 nm. For each detection, the total procedure was repeated 3 times.
Animal preparation and experimental design
All animal procedures were approved by the Committee on Animal Experimentation of Wuhan University, and the procedures were complied with the NIH Guide for the Care and Use of Laboratory Animals. Male BALB/c nude mice (4–5 weeks of age), obtained from the Center of Experimental Animals of Wuhan University, were used in all of the experiments. SW480, SW480/neo, and SW480/FADD cells, 5 × 106, suspended in 100 μl PBS, were subcutaneously inoculated into the lower right flank of the nude mice (n = 6 in each group). When the tumors were 100–150 mm3 in size, 5-fluorouracil (40 mg/kg) were administrated via intraperitoneal injection every 4 days. Tumor growth was monitored using calipers every 4 days. Tumor volume (V) was calculated by using the formula: tumor volume V (mm3) = π/6 × length (mm) × width (mm2). At the end of the experiment, tumors were harvested for additional analyses as described below. Differences in tumor growth were tested for statistical significance.
HE staining and TUNEL assay
Tumor tissues were fixed in 4% formaldehyde, dehydrated with gradient ethanol, and embedded in paraffin. Tissue sections (4 μm) were then dewaxed and rehydrated according to a standard protocol. For histologic analysis, sections were stained with hematoxylin and eosin (HE). For TUNEL assay, an in situ apoptosis detection kit (Roche Diagnostics, Branchburg, NJ, USA) was used to detect apoptotic cells in tumor tissue sections. Briefly, after incubation with proteinase K and rinsing with ddH2O, tumor sections were dewaxed with dimethylbenzene and rehydrated with gradient ethanol. Endogenous peroxidase was blocked with 3% H2O2, and sections were incubated with equilibration buffer and terminal deoxynucleotidyl transferase (TdT) enzyme. Finally, the sections were incubated with antidigoxigenin-peroxidase conjugate. Peroxidase activity in each section was shown by the application of DAB. Sections were counterstained with hematoxylin. The positive cells were identified, counted (three random fields per slides), and analyzed under the light microscopy (Carl Zeiss, Thornwood, NY, USA).
All data were expressed as Means ± S.E.M. The means of the different groups were compared using one-way ANOVA test. All statistical analyses were performed with the SPSS13.0 software (SPSS Inc., Chicago, IL, USA). Significant differences were accepted when P < 0.05.
Construction of eukaryotic vector of pEGFP-N1-FADD and overexpression of FADD in SW480 cells
To investigate the mRNA and protein expression of FADD in SW480, SW480/neo, and SW480/FADD cells, the levels of FADD were measured by RT-PCR assay and Western blotting assay. The relative expression of FADD to GAPDH or actin was shown. The mRNA and protein levels of FADD in SW480/FADD cells were significantly greater than that in SW480 cells and SW480/neo cells (P < 0.05). The brightness of FADD bands between SW480 cells and SW480/neo cells had no significant difference (P > 0.05) (Fig. 1C).
Cellular sensitivity to 5-fluorouracil
Overexpression of FADD enhances 5-fluorouracil-induced apoptosis and its mechanism in SW480 cells
Annexin V-FITC/PI staining flow cytometry was performed to further confirm overexpression of FADD enhances 5-fluorouracil-induced apoptosis. As shown in Fig. 3B, after treatment with 5-fluorouracil for 48 h, overexpression of FADD induced a higher percentage of cell apoptosis, 33.3 ± 4.5% (P < 0.05), whereas, a lower percentage of cell apoptosis was observed in SW480 cells and SW480/neo cells (13.9 ± 3.2% and 14.1 ± 3.4%, respectively).
In vivo study of combination of FADD and 5-fluorouracil suppression tumor
Our in vitro data clearly verified that stable transfection of FADD gene enhances apoptosis-inducing effects of 5-fluorouracil in SW480 cells via activation of the apoptosis pathway. The in vivo study was investigated in colorectal cancer xenografts. Tumor growth curves were depicted to compare the difference of the antitumor efficacy during the course of the experiments.
5-Fluorouracil is the cornerstone in first-line agent of chemotherapy for advanced and metastatic colorectal carcinoma, but its response rate is only 15% as monotherapy [4, 12, 13]. Today it is used in combination with other agents, such as irinotecan, oxaliplatin, capecitabine, bevacizumab, cetuximab, and panitumumab, as first-line or second-line therapy, has resulted in significant improvement in response rate, disease-free survival, and overall survival . In general, systemic treatment of 5-fluorouracil-based chemotherapy for colorectal carcinoma has two major forms : adjuvant therapy and palliative therapy. It has been reported that, 3-year disease-free survival and overall survival of adjuvant therapy, were 70–90% , but response rate and 1-year overall survival of palliative therapy, were only 40–60% .
Although 5-fluorouracil-based chemotherapy has underwent remarkable improvements with new chemotherapeutics combinations and with the access new targeted therapies , due to the side effects of 5-fluorouracil on normal cells and drug resistance of tumor cells, it has been a research focus on how to ameliorate the 5-fluorouracil effects in colorectal cancer treatment.
The realization that identification of molecules within the apoptotic pathway that will potentiate tumor chemosensitivity will enable optimization of chemotherapy for the treatment of colorectal cancer, by enhancing the therapeutic response to chemotherapy, intensified efforts for the development of novel modulators of the apoptotic threshold of colorectal cancer cells. Our results indicate that the FADD overexpressing of SW480 colorectal cancer cells had an enhanced sensitivity to chemotherapy, as demonstrated by the more apoptosis rates at same chemotherapy doses and time.
Overexpression of FADD induces apoptosis is in a Fas ligand-independent fashion death receptors pathway . The death receptors pathway, also named extrinsic pathway, is one kind of pathways of apoptosis. And the other kind of apoptosis pathway is called the mitochondria pathway (intrinsic pathway) . In the death receptor pathway, ligands such as TNF, FAS ligand, TRAIL, or TNFSF10 interact with their respective death receptors TNFR1, FAS, DR4, or DR5. These interactions ultimately lead to the recruitment of the FADD and the activation of the protease caspase-8, then activates caspase-3 and other downstream caspases, which results in a proteolytic cascade that gives rise to the cell death .
It has been reported  that the level of apoptosis induction was reduced in vitro and in vivo in human osteosarcoma cell lines when overexpression of the dominant-negative FADD, directly supports our findings. Moreover, the study of phosphorylated FADD is concerned. Keiji et al. [21, 22] found phosphorylated FADD-dependent activation of the JNK/caspase pathway plays an essential role in the mechanisms of amplifications of chemotherapy-induced apoptosis.
In this study, FADD gene was transfected into human colorectal cancer cell lines which induced its overexpression. We found that FADD overexpression could enhance the apoptosis-inducing effects of 5-fluorouracil, by TUNEL assay and annexin V-FITC/PI staining flow cytometry. These results are consistent with the reported by Micheau et al. , in which FADD could enhance apoptosis in colorectal cancer cells induced by cisplatin in vitro. Moreover, Western blotting assay was used to detect the level of protein expression of the procaspase-8, procaspase-3 and their cleaved forms. And it was found that stable transfection of FADD gene could increase the cellular activity levels of cleaved caspase-8 and cleaved caspase-3 after treating with 5-fluorouracil. This accords with the functional mechanisms of FADD which activates caspase-8 [24, 25] and other caspases, such as caspase-3 . At last, the results were also proved in vivo. HE staining and TUNEL assay of the subcutaneous tumor sections demonstrated that combination of FADD and 5-fluorouracil caused obvious cell death in tumor mass via apoptosis.
Cancer formation is often a multistep process and there may be point mutation, de-regulation or deletion of proto-oncogenes and anti-oncogenes which may be responsible for the development of cancer . The main advantage of gene therapy is transfer of a particular gene to a specific group of mammalian or tumor cells so that the desired effect will be localized and normal cells are spared . There is no doubt that chemo-gene therapy, which is a combination of gene transfer and chemotherapy, will play an important role in advanced cancers . It has been reported [30–32] that replication-deficient recombinant adenoviral vectors are predominantly used for colorectal cancer gene therapy. Choi et al.  found the combination of adenoviral-mediated IFN-β gene therapy and 5-fluorouracil resulted in tumor regression, apoptosis, and improved survival in an established liver metastases model of colorectal cancer. This approach may allow for an effective clinical application of this therapy and warrants additional investigation. Patients of colorectal cancer maybe treated with intra-tumoral injection of combination of adenovirus-FADD and 5-fluorouracil, which is one of forms that are transferred to the clinic or in in vivo situation.
According to the study of Li et al. , Bid is a specific proximal substrate of caspase-8 in the Fas apoptotic signaling pathway. While full-length Bid is localized in cytosol, truncated Bid (tBid) translocates to mitochondria and thus transduces apoptotic signals from cytoplasmic membrane to mitochondria. Our further studies are required to make clear that whether overexpression of FADD enhancing apoptosis-inducing effects of 5-fluorouracil on human colorectal cancer cells which are cross-talking activation of mitochondria pathway of apoptosis.
These results of the study provide a novel strategy to improve chemotherapeutic sensitivity in colorectal cancer patients and reduce their side effects, thus establishing a basis for further exploring the roles of FADD gene in apoptosis regulation of colorectal cancer.
This study was supported by a Grant from National Natural Science Foundation of China (NO.30471690).