Enhanced therapeutic effects for human pancreatic cancer by application K-ras and IGF-IR antisense oligodeoxynucleotides

Figure 1 Detection of K-ras point mutation at 12 codon and its style in pancreatic cancer cell line Patu8988 by PCR-SSP. 1: β-actin; 2: R1-R4 pairing (CGT mutation); 3: R2-R4 pairing (GTT mutation); 4: R3-R4 pairing (GAT mutation); M: 100 bp DNA marker.

Figure 2 Sequence analysis of pancreatic cancer cell line Patu8988 (A) and BXCP-3 (B). Patu8988 had K-ras point mutation at codon 12 and its style was GGT→GTT (indicated in the square). BXCP-3 was a K-ras gene wild type cell line with normal GGT at codon 12; No other mutation styles were found.

Figure 3 Each value represents the mean ± SD from triplicate determinations. A: Growth curves of Patu8988 cells treated with different concentrations of K-ras ASODN at different incubation times; B: Growth curves of Patu8988 cells treated with different concentrations of IGF-IR ASODN at different incubation times; C: Growth curves of Patu8988 cells treated with the combination treatment of 16 mg/L K-ras ASODN and 16 mg/L IGF-IR ASODN at different incubation times; D: The inhibition effect of the combination treatment with K-ras ASODN and IGF-IR ASODN on tumor growth in nude mice.

Figure 4 Apoptosis of Patu8988 cells detected by flow cytometry with Annexin V-FITC dual staining. A: Control group; B: IGF-IR ASODN group; C: K-ras ASODN group; D: K-ras ASODN + IGF-IR ASODN group; E: Apoptosis rate of Patu8988 cells for different groups. ^bP < 0.01, vs control group. Each value represents the mean ± SD from triplicate determinations.

Figure 5 mRNA expression of K-ras gene in Patu8988 cells at 48 h post-transfection. A: RT-PCR analysis for K-ras gene in different groups. The β-actin gene was included as internal control. 1: IGF-IR ASODN group; 2: K-ras ASODN + IGF-IR ASODN; 3: K-ras ASODN group; 4: Control group; B: K-ras products quantified relative to the internal control β-actin. ^aP < 0.05, vs control. Each value represents the mean ± SD from triplicate determinations.

Figure 6 mRNA expression of IGF-IR gene in Patu8988 cells at 48 h post-transfection. A: RT-PCR analysis for IGF-IR gene in different groups. The β-actin gene was included as internal control. 1: K-ras ASODN group; 2: K-ras ASODN + IGF-IR ASODN; 3: IGF-IR ASODN group; 4: Control group; B: IGF-IR products quantified relative to the internal control β-actin. ^aP < 0.05 vs control. Each value represents the mean ± SD from triplicate determinations.

Figure 7 Transmission electron microscopic appearance of Patu8988 cell. A: Normal Patu8988 cell with intact cell membrane and nuclear membrane (× 12 000); B: Patu8988 cell treated with K-ras ASODN + IGF-IR ASODN for 48 h. The margination of condensed chromatin and membrane-bounded apoptotic bodies are observed (× 15 000); C: Necrotic cell (× 15 000).