340774-69-8 br Quantitative real time PCR br The
Quantitative real-time PCR
The levels of Fas and caspase-8 mRNA in pancreatic cancer 340774-69-8 and nude mouse tumor tissues were detected by quantitative po-lymerase chain reaction (qPCR). Cell processing before measure-ment was the same as that used for western blotting. Total RNA was extracted with TRIzol in accordance with the manufacturer's in-structions. The OD260/OD280 was used to determine the purity and concentration of the RNA. cDNA was synthesized using a reverse transcription kit. According to the instructions of a real-time quantitative PCR sample kit, the following reaction condi-tions were used: 95 C for 5 min and 95 C for 30 s, annealing at 60 C for 30 s, and extension at 72 C for 30 s for a total of 45 cycles. qPCR assays were repeated three times, and mRNA levels were determined according to the 2-△△Ct method. The primers are shown in Table 1 (Invitrogen, USA).
Cocultured pancreatic cancer cells were fixed with Immunol Staining Fix Solution (Beyotime Biotechnology, China) for 30 min, and Enhanced Immunostaining Permeabilization Buffer (Beyotime Biotechnology, China) was added for 5 min at room temperature. After three washes with PBS, cancer cells were incubated with test solution from a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis assay kit (Beyotime Biotechnology, China). Animal model tumor tissue sections were first dewaxed and incubated with Proteinase K (20 mg/ml, Beyotime Biotechnology, China), and the remaining steps are the same as those used for cytology.
Statistical analyses were performed using SPSS 17.0 (SPSS, Inc., USA). Data are expressed as the mean ± standard deviation (SD).
Fig. 5. DNT cells promoted pancreatic cancer cell and tissue apoptosis. A, TUNEL assays showing that the Panc-1 þ DNT group had more apoptotic cells than did the other two groups. B, TUNEL assays showing that the SW1990 þ DNT group had more apoptotic cells than did the other two groups. C, TUNEL assays showing that the DNT cell group and gemcitabine group had more apoptotic tissues than did the blank control group in the animal model. D, Western blot analysis of cleaved caspase-3 in cells from the three groups. E, Western blot analysis of cleaved caspase-3 in tumor tissues from the three groups. ***P < 0.001.
Comparisons between 2 groups were assessed using Student's t-test. Statistically significant differences among several groups were assessed using variance analysis. P values < 0.05 indicated statisti-cal significance.
DNT cell expansion
According to cell counts, the concentration of cells per well was as high as 1.0 106 cells/ml. DNT cells were also investigated by flow cytometry. The DNT cell phenotype was CD3þCD4 CD8 with >90% purity (Fig. 1A and B).
DNT cells inhibited pancreatic cancer growth via the Fas pathway in vitro
To ascertain the role and mechanism of DNT cells in pancreatic cancer growth, we cocultured DNT cells and pancreatic cancer cells and analyzed the effect. Panc-1 and SW1990 cell growth was significantly inhibited by DNT cells. However, treatment with DcR3 reduced the antitumor effect of DNT cells. Moreover, DcR3 had no effect on Panc-1 and SW1990 cell growth (Fig. 2A and B). Further-more, the expression of Fas, caspase-8 and cleaved caspase-8 in the Panc-1 or SW1990 group, Panc-1 or SW1990 þ DNT group and Panc-1 or SW1990 þ DNT þ DcR3 group was detected by western blotting and qPCR. Western blotting results showed that the pro-tein levels of Fas, caspase-8 and cleaved caspase-8 were higher in the Panc-1 or SW1990 þ DNT group than in the Panc-1 or SW1990 group, but these levels were decreased when blocking agent DcR3
was added (Fig. 2C). qPCR results revealed that Fas and caspase-8 mRNA levels in the three groups were similar to their protein levels (Fig. 2D and E).
DNT cells decreased the tumor size in animal models
DNT cells inhibited pancreatic cancer growth via the Fas pathway in vivo
To identify the mechanism of DNT cell cytotoxicity in vivo, we used western blotting and qPCR to determine Fas, caspase-8 and cleaved caspase-8 expression. The protein levels of Fas, caspase-8 and cleaved caspase-8 in the DNT cell group were higher than those in the blank control group, and no significant difference was observed between the DNT cell group and gemcitabine group (Fig. 4A). Additionally, the mRNA levels of Fas and caspase-8 in the three groups were the same as the protein levels (Fig. 4B).
DNT cells promoted pancreatic cancer cell and tissue apoptosis
To evaluate whether apoptosis occurred in pancreatic cancer after treatment with DNT cells, we performed a TUNEL apoptosis assay and examined the expression of cleaved caspase-3. The activation of caspase-3 is a biochemical hallmark of apoptosis. Our TUNEL assay demonstrated that the cancer cell þ DNT group had more apoptotic cancer cells than did the other two groups (Fig. 5A