The effect of adenovirus expressing wild-type p53 on 5-fluorouracil chemosensitivity is related to p53 status in pancreatic cancer cell lines

Abstract

Aim: There are conflicting data about p53 function on cellular sensitivity to the cytotoxic action of 5-fluorouracil (5-FU). Therefore the objective of this study was to determine the combined effects of adenovirus-mediated wild-type (wt) p53 gene transfer and 5-FU chemotherapy on pancreatic cancer cells with different p53 gene status.

Methods: Human pancreatic cancer cell lines Capan-1(p53mut), Capan-2(p53wt), FAMPAC(p53mut), PANC1(p53mut), and rat pancreatic cancer cell lines AS(p53wt) and DSL6A(p53null) were used for in vitro studies. Following infection with different ratios of Ad-p53-particles (MOI) in combination with 5-FU, proliferation of tumor cells and apoptosis were quantified by cell proliferation assay (WST-1) and FACS (PI-staining). In addition, DSL6A syngeneic pancreatic tumor cells were inoculated subcutaneously in to Lewis rats for in vivo studies. Tumor size, apoptosis (TUNEL) and survival were determined.

Results: Ad-p53 gene transfer combined with 5-FU significantly inhibited tumor cell proliferation and substantially enhanced apoptosis in all four cell lines with an alteration in the p53 gene compared to those two cell lines containing wt-p53. In vivo experiments showed the most effective tumor regression in animals treated with Ad-p53 plus 5-FU. Both in vitro and in vivo analyses revealed that a sublethal dose of Ad-p53 augmented the apoptotic response induced by 5-FU.

Conclusion: Our results suggest that Ad-p53 may synergistically enhance 5-FU-chemosensitivity most strikingly in pancreatic cancer cells lacking p53 function. These findings illustrate that the anticancer efficacy of this combination treatment is dependent on the p53 gene status of the target tumor cells.

Figure 1

Expression of p53 protein before (-) and after infec-tion with Ad-p53 (+) in pancreatic cancer cell lines.

Figure 2

Decreased in vitro growth rate in pancreatic cancer cell lines 24 h after infection with Ad-p53. Data points repre-sent means from 9 samples of viable cells in three indepen-dent experiments; bars, SD.

Figure 3

Percentage of apoptotic cells of human and rat pan-creatic carcinoma cell lines either infected with Ad-CD con-trol vector or Ad-p53 (MOI 1) 24 h prior ± 5-FU chemotherapy (5 µg/mL) given for 48 h. Points represent means from 9 samples in three independent experiments; bars, 95%CI.

Figure 4

Gel electrophoresis for PCR products from DSL6A tumors removed from Lewis rats 24 h following the last treatment. (P) positive control, vector DNA; (N) negative con-trol (RT minus control); (C) Ad-CD control virus treated tu-mor (Ad-CD); (T) Ad-p53 infected tumor.

Figure 5

Efficacy of Ad-p53 infection and 5-FU as single agents vs the combination of both on tumor growth of Lewis rats challenged with DSL6A tumor cells. Arrowheads indicate treat-ment application. ¹All animals were killed after 6 cycles of therapy because the tumor size was above 600 mm².

Figure 6

Ad-p53 mediated sensitisation of tumors to apoptosis in response to 5-FU in vivo. Formalin-fixed tissue sections were analyzed by TUNEL labelling. Original magnification × 200.