Up-regulation of integrin beta3 in radioresistant pancreatic cancer impairs adenovirus-mediated gene therapy

Abstract

Adenovirus-mediated gene therapy is a promising approach for the treatment of pancreatic cancer. We previously reported that radiation enhanced adenovirus-mediated gene expression in pancreatic cancer, suggesting that adenoviral gene therapy might be more effective in radioresistant pancreatic cancer cells. In the present study, we compared the transduction efficiency of adenovirus-delivered genes in radiosensitive and radioresistant cells, and investigated the underlying mechanisms. We used an adenovirus expressing the hepatocyte growth factor antagonist, NK4 (Ad-NK4), as a representative gene therapy. We established two radioresistant human pancreatic cancer cell lines using fractionated irradiation. Radiosensitive and radioresistant pancreatic cancer cells were infected with Ad-NK4, and NK4 levels in the cells were measured. In order to investigate the mechanisms responsible for the differences in the transduction efficiency between these cells, we measured expression of the genes mediating adenovirus infection and endocytosis. The results revealed that NK4 levels in radioresistant cells were significantly lower (P < 0.01) than those in radiosensitive cells, although there were no significant differences in adenovirus uptake between radiosensitive cells and radioresistant cells. Integrin beta3 was up-regulated and the Coxsackie virus and adenovirus receptor was down-regulated in radioresistant cells, and inhibition of integrin beta3 promoted adenovirus gene transfer. These results suggest that inhibition of integrin beta3 in radioresistant pancreatic cancer cells could enhance adenovirus-mediated gene therapy.

Figure 1

NK4 expression in Ad‐NK4‐treated radioresistant pancreatic cancer cells was much lower than that in Ad‐NK4‐treated radiosensitive pancreatic cancer cells. (a) CFPAC‐1 parent cells, and two established radioresistant pancreatic cancer cells (R1, R2) were plated and irradiated with 2 Gy or 5 Gy. Their survival was determined by propidium iodide assay 72 h after radiation, and defined as the ratio relative to unirradiated cells. Each value represents the mean ± SD of three independent samples. *P < 0.05. (b) Cells were infected with Ad‐NK4 at 10 multiplicities of infection (MOI) and proteins were isolated on post‐infection day 2. NK4 concentrations were determined by enzyme‐linked immunosorbent assay (ELISA) and defined as the ratio relative to radiosensitive cells (CFPAC‐1 parent cells). Each value represents the mean ± SD of three independent samples. **P < 0.01. (c) Cells were infected with Ad‐NK4 at 10 MOI and NK4 levels in the culture media were measured by ELISA on post‐infection day 2 and defined as the ratio relative to radiosensitive cells (CFPAC‐1 parent cells). Each value represents the mean ± SD of three independent samples. **P < 0.01.

Figure 2

β‐Galactosidase (β‐gal) expression in Ad‐lacZ‐treated radioresistant cells was lower than that in Ad‐lacZ‐treated radiosensitive cells, but there were no differences in adenovirus uptake between radioresistant and radiosensitive pancreatic cells. Cells were infected with Ad‐lacZ at 10 multiplicities of infection (MOI) and β‐gal activity was assessed by X‐gal staining at 48 h after infection. (A) Photomicrographs of X‐gal‐stained radiosensitive or radioresistant cells, ×100. a, CFPAC‐1 parent cells; b, R1 cells; c, R2 cells. (B) Number of β‐gal‐positive cells. Each value represents the mean ± SD of five independent fields. **P < 0.01. (C) Cells were infected with Ad‐lacZ at 10 MOI and DNA was extracted at 24 h after infection. Viral DNA content was quantified by real‐time PCR and defined as the ratio compared with radiosensitive cells. Each value represents the mean ± SD of triplicate measurements.

Figure 3

Lower expression of Coxsackie virus and adenovirus receptor (CAR) and much higher expression of integrin β3 in radioresistant cells were associated with decreased adenovirus gene expression (A) (a) CAR and (b) integrin β3 mRNA expression in cells. CAR and integrin β3 mRNA were quantified by real‐time RT‐PCR from total RNA from radiosensitive cells (CFPAC‐1 parent cells) or radioresistant cells (R1, R2) and defined as the ratio relative to radiosensitive cells. (B and C) Inhibition of integrin β3 mRNA expression by siRNA‐recovered NK4 expression in radioresistant cells. (B) Integrin β3 mRNA was quantified by real‐time RT‐PCR from total cellular RNA from integrin β3‐specific siRNA‐transfected cells or control siRNA‐transfected cells at 24, 48, and 72 h after transfection and was expressed as fold‐decrease compared with control siRNA cells. Each value represents the mean ± SD of triplicate measurements. (C) Radioresistant cells were transfected with integrin β3‐specific siRNA or control siRNA and infected with Ad‐NK4 at 10 multiplicities of infection at 48 h after transfection. NK4 levels in the culture media were measured by enzyme‐linked immunosorbent assay and expressed as fold‐increase compared with control siRNA cells. Each value represents the mean ± SD of three independent samples. *P < 0.05.