Figure 5

Cooperative effects of the combination treatment of CEBPD and granzyme B in PC3 and LNCaP cells. (a) Scheme of the protease-activated fusion protein. The diagram illustrates the design of the prodrugs PF-CEBPD and PF-granzyme B. PF can form a pore structure at the cell membrane and the protease substrates (PSA, HSSKLQ; MMP2, GPLGVR) can be cleaved by the proteases PSA or MMP2. Once the proteases act, the relaxed CEBPD or granzyme B can enter the cancer cell through the pore formed by PF. L: leader sequence of a murine Ig kappa chain. Myc: c-myc epitope. His: 6x histidine tag. (b) The detection of prodrugs in the supernatant of stably expressing PF-CEBPD and PF-granzyme B 3T3 cells. To generate stable cell lines, all plasmids were co-transfected with pVSVG in GP2-293 cells (Clontech, BD Biosciences, Taiwan) to produce recombinant retroviral particles. After 48 h of transfection, the culture medium was mixed with 8 μg/ml polybrene and added to 3T3 cells. Permanent cells were selected with 0.1 mg/ml Hygromycin B and sorted for high surface expression to produce stable cells. The expression of protease-specific fusion proteins (PSA-CD, PSA-GB, MMP2-CD and MMP2-GB) in 3T3 cells was detected by western blot analysis using an anti-c-myc antibody. (c) The combination of prodrugs of granzyme B and CEBPD enhances CASP8 and CASP3 cleavage. LNCaP cells or PC3 cells were plated in serum-free medium in microtiter plates. After 48 h, the LNCaP cells were treated with the culture supernatants of PSA-GB, PSA-CD or a mixture of PSA-GB and PSA-CD. PC3 cells were treated with the supernatants of MMP2-GB, MMP2-CD or mixture of MMP2-GB and MMP2-CD in triplicate. The cellular activity of CASP8 or CASP3 was measured via the Caspase-Glo 8 or Caspase-Glo 3 Assay Systems. Columns, the average of three independent measurements in triplicate; bars, mean±S.D. (***P<0.001; Student’s t-test). (d) The combination of prodrugs of granzyme B and CEBPD represses PrCa cell proliferation. LNCaP cells were treated with the culture supernatants of PSA-GB, PSA-CD or a mixture of PSA-GB and PSA-CD. PC3 cells were treated with the culture supernatants of MMP2-GB, MMP2-CD or mixture of PSA-GB and PSA-CD. The cell viability was determined using the ATPlite kit (Perkin-Elmer, Waltham, MA, USA). Columns, the average of three independent measurements in triplicate; bars, mean±S.D. (***P<0.001; Student’s t-test)