Figure 4.

C-26 cells overexpressing HO-1 are less sensitive to PDT treatment both in vitro and in vivo. (a) C-26 cells were stably transfected with a control plasmid (C-26-pcDNA3) or plasmid containing HO-1 (C-26-B6). The bars represent expression level of HO-1 in cell lysates of C-26-pcDNA3 cells and C-26-B6 cells measured by ELISA and expressed as mean concentration (pg/ml)±s.d. (b) C-26-pcDNA3 and C-26-B6 cells were exposed to 10 μg/ml Photofrin for 24 h and then to 6.0 kJ/m² light and incubated for 8 h. Total cell lysates were prepared, and Western blot analysis was performed using anti-HO-1 or anti-α-tubulin antibodies. (c) C-26, C-26-pcDNA3 and C-26-B6 cells were seeded onto 35 mm plates at the concentration of 2.5 × 10⁵ cells/3 ml/dish, incubated for 24 h with 10 μg/ml Photofrin and then exposed to different doses of light, from 3 to 12 kJ/m². Immediately after PDT, cells were trypsinized and seeded onto 96-well plate in the dilution of 1:50 in eight repeats. Following 24 h of incubation, the cytotoxic effects were measured by crystal violet staining. The bars represent percent cytotoxicity versus untreated controls, different for each separate cell line. Data refer to mean±s.d. * P<0.05 versus nontransfected controls and C-26 cells transfected with empty vector (Student's t-test). (d) BALB/c mice were inoculated with 1 × 10⁶ C-26, C-26-pcDNA3 or C-26-B6 cells into the right hindlimb. Photofrin™ was administered intraperitoneally at a dose of 10 mg/kg on fifth day after inoculation of tumor cells and 24 h later, the tumor site was illuminated with a laser light at a dose of 120 J/m². Measurements of tumor diameter were started on fifth day of the experiment and were performed every 2 days. The graph represents the influence of the PDT treatment on the growth of C-26 tumors (n = 8–9).