The study included 68 patients with primary GC (mean age 55 years; range 33–75 years; male: female, 40:28) and was approved by the local ethics committee. The patients underwent gastrectomy at the General Hospital of Jinan Military Command from 2007 Jan to 2008 Dec. After surgery, the tumor specimens and distant normal gastric mucosa tissues were collected for this study. None of the patients enrolled in the study had received chemotherapy or radiotherapy prior to surgery, and there was no evidence of any other malignancies. The diagnoses of all GCs were histopathologically confirmed by examination of surgical specimens. The clinical stage of the patients and the pathologic grade of tumors were determined according to the TNM classification and WHO criteria, respectively. The stage was IA in 4 (5.9%), IB in 14 (20.6%), II in 23 (33.8%), IIIA in 15 (22.1%), IIIB in 6 (8.8%), IV in 6 (8.8%). There were 13 (19.1%) cases of GC with well differentiated, 20 (29.4%) cases with moderately differentiated, 35 (51.5%) cases with poorly differentiated. Furthermore, chronic superficial gastritis tissues (n=6) obtained from patients which underwent gastroscopic biopsy without GC were studied.
The anticancer drugs tested contained Taxol (TAX) (Taiji Co. Let., Sichuang, China), Cisplatin (CDDP) (Qilu Co. Let., Shandong, China), 5-Fluorouracil (5-FU) (Hualian Co. Let., Shanghai, China), Adriamycin (ADM) (Xinhua Co. Let., Shandong, China), and Mitomycin (MMC) (Huangshi Co. Let., Hubei, China). Each drug was diluted in a complete medium at 10-fold therapeutic peak plasma concentration as reported previously . The complete medium consisted of RPMI 1640 (Gibco Gaithersburg, MD, USA) supplemented with 10% heat-inactivated calf serum (Gibco), 2 mM L-glutamine, and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin).
MTT chemosensitivity assay
The in vitro chemosensitivity of fresh surgical specimens of GC was evaluated using the MTT assay as reported by Mossman, with some modifications . The tissue specimens obtained during surgery were from patients who had given written informed consent. Resected specimens were stored in Hank’s balanced salt solution (Gibco Gaithersburg, MD, USA) that contained 100 IU penicillin, 100 μg streptomycin and 0.25 μg amphotericin B (all from Gibco) per ml. Single-cell suspensions were prepared enzymatically by incubating the specimens for 30 min in 0.5 mg/ml pronase, 0.2 mg/ml collagenase type І and 0.2 mg/ml DNase (all from Sigma). After 2 centrifugations (1000 r/min), the tumor cells were suspended in RPMI 1640 medium supplemented with 10% fetal bovine serum, diluted to 1×105 cells/ml and 100 μl aliquots were plated into 96 well microplates (Gibco) to give approximately 104 cells per well. The drug solutions were dissolved in RPMI 1640 and 100 μl aliquots were added to each well to give final concentrations of 10 μg/ml MMC, 50 μg/ml 5-FU, 25 μg/ml CDDP, 5 μg/ml TAX, or 4 μg/ml ADM. The control wells contained 100 μl of the cell suspension and 100 μl RPMI 1640 containing 10% FBS, and 200 μl RPMI with 10% FBS was used as a blank. The plates were incubated for 48 h at 37°C in a humidified atmosphere containing 95% air and 5% CO2. 20 μl mixture of 0.4% MTT (Sigma) and 0.1 M sodium succinate (each dissolved in 10 μl phosphate-buffered saline and filtered through a 0.45 μm membrane filter (Millipore, Bedford, MA, USA)), was then added and the plates were incubated for an additional 3 h at 37°C. After the final incubation, 150 μl dimethyl sulfoxide (Gibco) was added to each well to dissolve the MTT-formazan salt and the plates were mechanically shaken for 10 min on a mixer. The optical densities of each well were determined using a model SM-3 easy reader (Tianshi, Beijing, China) at 570 nm. The inhibition rates (IR) were calculated using the formula (1 – A/B) ×100%, where A and B represent the mean absorbance of the drug-treated and control wells, respectively. The effects were regarded as positive when IR values were ≥ 30%.
In situ hybridization (ISH) was carried out by using an hTERT ISH detection kit (produced by Wuhan Boster Biological echnology Ltd.). The antisense poly-oligonucleotide probe was digoxin-labeled. Formalin-fixed, paraffin-embedded samples were cut at 5 μm and adhered to poly-l-lysine treated slides. Samples were deparaffinized and rehydrated through a graded series of ethanol, and endogenous peroxidase was blocked using 3% hydrogen peroxide for 10 min. The slides were digested with pepsin at 37°C for 15–20 min. 20 μl of probe was hybridized to each slide for 16–20 h at 40°C. After hybridization, excess probe was removed by washing in 2×SSC at 37°C. Tissue sections were reblocked for 20 min with blocking reagent, and then the primary antibody (rabbit anti-digoxin antibody) was added for 60 min at 37°C. After washing with 0.5 M PBS three times at 5 min each, the slides were incubated with the secondary goat anti-rabbit immunoglobulin (IgG) antibody conjugated with biotin for 20 min at 37°C, then washed with 0.5 M PBS again as previously described. Samples were next incubated with SABC for 20 min at room temperature and rinsed with 0.5 M PBS for four times at 5 min each. The reaction products of peroxidase were visualized by incubation with chromogen diaminobenzidine for 15–20 min. Finally, the slides were counterstained for nuclei by haematoxylin stain. A negative control was prepared for each sample using a hybridization solution without probe. The positive signals of hTERT mRNA expression were stained with the color of brown-yellow located in cell plasma. The average percentage of positive cells was determined in at least 5 areas at ×400 and assigned to one of four categories: (−)-negative or equivocal staining; (+)-weak positive, cells were stained in 1-25%; (++)-middle positive, cells were stained in 25-50%; and (+++)-strong positive expression, cells were stained over 50%.
Quantitative results were expressed as mean ± standard error of mean. Significant differences were determined by Fisher’s PLSD test or a Chi-square test. The associations analysis were tested with Spearman’s test for nonparametric correlation. A P value of less than 0.05 was considered to be statistically significant.