Immunohistochemical evaluation of TS and DPD in UTUC human samples
Surgical specimens from 176 patients who had been surgically treated for UTUC at Keio University Hospital from 1986 to 2007 were examined. The median follow-up was 45 months and the median patient age was 67 years (range, 36–89 years). The patients did not undergo any chemotherapy or radiation therapy prior to the surgery. Patients with distant metastasis at the time of diagnosis and incomplete clinical data were excluded from the study. A nephroureterectomy with the removal of the bladder cuff was the most common procedure (n = 172), while a partial ureterectomy was performed in the remaining 4 patients. Regional lymphadenectomy was generally performed in patients with suspicious lymph nodes on preoperative axial imaging or with adenopathy detected during an intraoperative examination. Extended lymphadenectomy was not routinely performed. The patients were followed postoperatively with urinary cytology every 3 months for 2 years and every 6 months thereafter. Computed tomography as well as cystoscopy and magnetic resonance imaging were performed every 6 months for 5 years and annually thereafter.
Tissue samples were obtained from consented patients in this study which was approved by Keio University Ethics Committee. All specimens were fixed in 10% formalin and embedded in paraffin, and all slides were re-reviewed by genitourinary pathologists. Tumors were staged according to the American Joint Committee on Cancer-Union Internationale Contre le Cancer TNM classification. Tumor grading was assessed according to the 1998 WHO/International Society of Urology Pathology consensus classification
. Lymphovascular invasion (LVI) was defined as the presence of tumor cells within an endothelium-lined space without underlying muscular walls.
Sections (4 μm) of formalin-fixed and paraffin-embedded material were analyzed. The sections were deparaffinized in xylene and rehydrated in graded alcohols and distilled water. After antigen retrieval with citric acid (pH 6.0), endogenous peroxidase activity was blocked with 1% hydrogen peroxide for 30 minutes followed by washing with distilled water. To bind nonspecific antigens, the sections were incubated with 5% skim milk for 15 minutes. The sections were incubated with either an anti-TS rabbit monoclonal antibody (1:100 dilution, Taiho Pharmaceutical Co, Tokyo, Japan) at room temperature for 1 hour or an anti-DPD mouse polyclonal antibody (1:100 dilution, Taiho Pharmaceutical Co.) at room temperature for 1 hour. After washing with phosphate-buffered saline (PBS), they were incubated with secondary antibodies against rabbit IgG conjugated to a peroxidase-labeled dextran polymer (no dilution, anti-rabbit Envision, Dako Japan, Tokyo) or against mouse IgG conjugated to a peroxidase-labeled dextran polymer (no dilution, anti-mouse Envision, Dako Japan) for 1 hour. Color was developed with 3,3’-diaminobenzamine tetrahydrochloride in 50 nmol/L Tris–HCl (pH 7.5) containing 0.005% hydrogen peroxidase. The sections were counterstained with hematoxylin.
To evaluate TS and DPD staining, cancer cells with positive staining in the cytoplasm were counted in at least 10 representative fields selected randomly by a uro-pathologist and staining intensity that was stratified from 0 to 3 (0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining) was estimated. Cases with more than 25% positive tumor cells (moderate and strong staining) in a section were regarded as positive expression as previously described
. The evaluation of immunostaining was made by a uro-pathologist who was unaware of the clinico-pathological data and clinical outcomes of the patients.
Cell lines and chemicals
Three human UC cell lines, T24, 5637, and UMUC-3 (American Type Culture Collection, Rockville, MD, USA), were grown in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 100 μg/ml streptomycin (Life Technologies, Inc., Grand Island, NY, USA), and 100 IU/ml penicillin (Life Technologies, Inc., Grand Island, NY, USA).
5-FU was synthesized by Wako Pure Chemical Industries (Osaka, Japan) and CDHP, tegafur, UFT and S-1 were kindly supplied by Taiho Pharmaceutical Co., Ltd (Tokyo, Japan). 5-FU and CDHP were dissolved in culture medium to prepare a 100 μg/ml solution and subsequently diluted in culture medium. Tegafur, UFT and S-1 were dissolved in distilled water with 0.5% hydroxypropylmethylcellulose (HPMC).
Cell growth assay
Briefly, 2 × 104 cells were seeded into each well of 96-well plates and allowed to grow overnight. The cells were then treated with various concentrations of 5-FU with or without CDHP. After 72 hours of incubation, cytotoxicity was determined using WST-1; 4-[3-(4-lodophenyl)-2-(4-nitrophenyl)-2 H-5-tetrazolio]-1,3-benzene disulfonate (Takara Bio Inc, Shiga, Japan). The absorbance value of each well was determined at 450 nm with a 650 nm reference beam by a microplate reader (Bio-Rad Laboratories, Inc, Tokyo, Japan). As previously described
, the 5-FU concentration causing 50% growth inhibition compared with the control (IC50) was calculated from the regression line. These experiments were repeatedly performed at different days, where new cells were grown.
Enzyme-linked immunosorbent assasy (ELISA)
Each sample (1.0 × 108 cells or xenograft tumors) was homogenized in a 10-fold volume of sample weight of the diluting solution (20 mM PBS which contained 0.05% Tween 20) and centrifuged at 105,000× g, 4°C for an hour. The supernatant (100 μl) was then dispensed onto an anti-human TS or DPD polyclonal antibody (Solid phase antibody: Mitsubishi Chemical Medience, Tokyo, Japan) immobilized plate and incubated for an hour at room temperature. After the wells were washed four times with PBS, 100 μl aliquots of horseradish peroxidase were conjugated to anti-human TS or DPD polyclonal antibody (Label antibody: Mitsubishi Chemical Medience, Tokyo, Japan). After the wells were washed four times with PBS, 100 μl aliquots of 0.1 M acetate buffer (pH 5.5; color-developing solution) containing 3 mg/ml orthphenylenediamine and 0.75 mM hydrogen peroxide were added, followed by incubation for 30 minutes in the dark. Finally, 100 μl aliquots of 1 M sulfuric acid were added to terminate the reaction and the measurements were conducted with the measuring wavelength of an ELISA plate reader set at 490 nm.
Real-time quantitative PCR (RT-PCR)
The cells were lysed with RNAiso reagent (Takara Bio Inc, Shiga, Japan) according to the manufacturer’s directions for total RNA extraction. RNA was quantitated by the ratio of absorbance at 260/280 nm. Reverse transcription of RNA to cDNA was carried out using a High Capacity cDNA Archive Kit (Applied Biosystems, Tokyo, Japan). Next, real time PCR was carried out in a final volume of 20 μl containing cDNA template, TS, DPD or GAPDH primers (Applied Biosystems, Tokyo, Japan) and TaqMan® Universal PCR Master Mix (Applied Biosystems, Tokyo, Japan), and DNase RNAase free water, using a StepOne real time PCR system (Applied Biosystems, Tokyo, Japan) according to the manufacturer’s protocol. Cycling conditions were 50°C for 10 minutes, 95°C for 10 minutes, and then 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. The data were then quantified using the comparative C
t method for relative gene expression compared with GAPDH as endogenous control. The primers and TaqMan probe sets for TS (TYMS) (Hs00426591_m1), DPD (DPYD) (Hs00559279_m1), and human GAPDH endogenous control (Hs99999905_m1) were purchased from Applied Biosystems.
Small interfering RNA (siRNA)
Three predesigned siRNAs for DPYD, TYMS and nontargeting control (NTC) siRNA (AllStars Negative Control siRNA) were obtained from Invitrogen Co (Tokyo, Japan). UMUC-3 cells (1.5 × 105 per well) were cultured in antibiotic-free medium overnight at 37°C in 5% CO2 and then transfected with each siRNA for DPYD or TYMS or nontargeting control, using Lipofectamine Max (Invitrogen Co, Tokyo, Japan). Forty-eight hours later, the transfected cells were washed and used for subsequent experiments.
Treatment in vivo
All of the procedures involving animals and their care in this study were approved by the Animal Care Committee of Keio University in accordance with institutional and Japanese government guidelines for animal experiments. Female BALB/c-nu/nu mice were obtained from Sankyo Lab Service Co. (Tokyo, Japan). UMUC-3 cells (2 × 106) were implanted subcutaneously into the flank of each nude mouse. When a mouse developed a palpable tumor, it was randomly assigned to one of 4 groups. Treatment groups were dosed at the maximal tolerable dose; tegafur 180 mg/kg/day, UFT 20 mg/kg/day, and S-1 8.3 mg/kg/day
. Each drug was administered daily by gavage. Control animals only received vehicle by gavage. Each experimental group consisted of 10 mice. The mice were carefully monitored, and tumor size and body weight were measured every 4 days. Tumor volume was calculated according to the formula a2 × b × 0.52, where a and b are the smallest and largest diameters, respectively. Eight weeks after tumor cell implantation, the mice were sacrificed and the tumors were collected. The levels of TS and DPD in the tumors were then measured by ELISA as described previously.
The association between TS and DPD, and clinico-pathological features was assessed using χ2 test. Disease-specific survival (DSS) and progression-free survival (PFS) were calculated by the Kaplan-Meier method and analyzed by the log-rank test. Cox proportional hazards regression analysis was used to assess the prognostic indicators that included age, gender, tumor location, pT, grade, nodal involvement, LVI, TS expression, and DPD expression for survival. The difference between two groups in in vitro study and in the animal model was assessed with the Mann–Whitney U-test. The level of statistical significance was set at P <0.05. These analyses were performed with an SPSS Version 16.0 statistical software package (SPSS Corporation).