Dickkopf3 overexpression inhibits pancreatic cancer cell growth in vitro

Figure 1 Dickkopf3 expression in human pancreatic cancer cell lines (PANC-1, MIA PaCa-2, AsPC-1 and BxPC-3). A, B: Dickkopf3 (Dkk3) mRNA expression was detected by reverse transcription polymerase chain reaction (RT-PCR) and real-time RT-PCR. Dkk3 mRNA expression was low in all cell lines examined. Dkk3: Dickkopf3; RQ: Relative quantitation.

Figure 2 Dickkopf3 promoter methylation analysis in human pancreatic cancer cell lines. A: Methylation-specific PCR (MSP) was performed with bisulfite-treated DNA from pancreatic cancer cells. The Dickkopf3 (Dkk3) promoter was significantly methylated in MIA PaCa-2 and AsPC-1 cells; B: MSP controls demonstrate the specificity of the Dkk3 primers used. Methylated bisulfite-converted DNA exclusively yields amplification products with primers specific to methylated Dkk3 promoter sequences; unmethylated bisulfite-converted DNA yields exclusively amplification products with primers recognizing unmethylated Dkk3 promoter sequences. MD: Methylated bisulfite-converted DNA; UD: Unmethylated bisulfite-converted DNA; U: PCR products amplified with primers recognizing unmethylated Dkk3 promoter sequences; M: Amplification generated with methylation-specific primers.

Figure 3 Dickkopf3 mRNA expression after demethylation in vitro. A, B: AsPC-1 and MIA PaCa-2 cells were treated with 10 μmol/L and 20 μmol/L of the DNA demethylating agent, 5-aza-dC, for 48 h or 72 h, respectively. The results show that these two cell lines, in which the Dickkopf3 (Dkk3) promoter was initially heavily methylated, had increased Dkk3 mRNA expression that was dependent on the dosage and timing of 5-aza-dC treatment (^aP < 0.05 vs untreated MIA PaCa-2 cells or untreated AsPC-1 cells, ^bP < 0.01 vs untreated MIA PaCa-2 cells or untreated AsPC-1 cells); C, D: Control cells were incubated with dimethyl sulfoxide and fresh medium. RQ: Relative quantitation; Dkk3: Dickkopf3; DMSO: Dimethyl sulfoxide.

Figure 4 The effects of dickkopf3 overexpression on pancreatic cancer cells. MIA PaCa-2 cells were transfected with pcDNA3.1-dickkopf3 (Dkk3) or pcDNA3.1 vector. A, B: After transfection, Dkk3 mRNA and protein expression were examined by real-time reverse transcription polymerase chain reaction (real-time RT-PCR) and Western blotting. The results show that in the pcDNA3.1-Dkk3-transfected MIA PaCa-2 cells, Dkk3 expression was significantly upregulated (P < 0.01, ^aP < 0.01 vs MIA PaCa-2 cells). B, C: β-catenin expression was examined by real-time RT-PCR and western blotting. β-catenin expression was downregulated 48 h and 72 h after transfecting pcDNA3.1-Dkk3 into MIA PaCa-2 cells (P < 0.01, ^aP < 0.01 vs MIA PaCa-2 cells). B: The expression of extracellular signal-regulated protein kinases (ERK) and phosphorylated extracellular signal-regulated protein kinases (pERK) was examined by western blotting. The expression of pERK was simultaneously downregulated, without a significant change in total ERK expression. D: MTS assay results showed that the proliferative ability of pcDNA3.1-Dkk3-transfected cells was lower than that of pcDNA3.1-transfected cells (P < 0.01, ^aP < 0.01). E: Dose-response analysis of pcDNA3.1- or pcDNA3.1-Dkk3-transfected MIA PaCa-2 cells with gemcitabine treatment. Seventy-two hours after gemcitabine addition, the IC50 values for gemcitabine were 0.621 μmol/L for pcDNA3.1-Dkk3-transfected cells and 1.877 μmol/L for pcDNA3.1-transfected cells. PERK: Phosphorylated extracellular signal-regulated protein kinases; ERK: Extracellular signal-regulated protein kinases; RQ: Relative quantitation; Dkk3: Dickkopf3; DMSO: Dimethyl sulfoxide.