Missing Selectivity of Targeted 4β-Phorbol Prodrugs Expected to be Potential Chemotherapeutics

Abstract

Targeting cytotoxic 4β-phorbol esters toward cancer tissue was attempted by conjugating a 4β-pborbol derivative with substrates for the proteases prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) expressed in cancer tissue. The hydrophilic peptide moiety was hypothesized to prevent penetration of the prodrugs into cells and prevent interaction with PKC. Cleavage of the peptide in cancer tumors was envisioned to release lipophilic cytotoxins, which subsequently penetrate into cancer cells. The 4β-phorbol esters were prepared from 4β-phorbol isolated from Croton tiglium seeds, while the peptides were prepared by solid-phase synthesis. Cellular assays revealed activation of PKC by the prodrugs and efficient killing of both peptidase positive as well as peptidase negative cells. Consequently no selectivity for enzyme expressing cells was found.

Figure 1

Target compounds and starting material: 4β-Phorbol 12-O-myristate 13-O-acetate (1), toxin 2 obtained after cleavage of prodrugs 4 and 5 with hK2 or PSA, respectively, while toxin 3 is obtained after cleavage of prodrug 6 with PSMA. 4β-Phorbol (7). Compound 8 is the starting material for synthesis of compounds 2–3, and compound 9 for 4–6.

Figure 2

Displacement binding curves of prodrugs 4–6. Toxins 2 and 3 and PMA. Binding of [³H]PDBu (10 nM) to PKCα was measured in the presence of increasing concentrations of the tested compounds. The PKCα was obtained from a lysate if cells overexpressed the enzyme. The data is presented as mean of residual [³H]PDBu binding (% of control) from three parallel samples in a single representative experiment.

Figure 3

Effects of phorbol prodrugs 4–6 and cleavage products 2 and 3 as well as Tg3 and Tg6 on viability of PCa cell lines. (A) PC3; (B) LNCaP; (C) DU145; (D) 22Rv1; and the effect of PKC inhibitor Gö6983 (1 μM) on the effect of 20 μM of 2–6, Tg3, Tg6, and 100 nM PMA on the viability of LNCaP, 22Rv1, DU145, and PC3 cells (E). Cell viability was measured after 72 h incubation with the compounds by utilizing the MTT assay. The data is presented as mean of cell viability (% of control) (n = 3).

Figure 4

Effects of phorbol derivatives 2–6 on ERK1/2 phosphorylation in PC cells. Quantifications from DU145 and 22Rv1 cells. Data is presented as mean + SEM (N = 3; *P < 0.05 vs ctrl, Welch’s t test). The cells were treated with 20 μM of different phorbol derivatives and PMA for 30 min. The cells were harvested, and then ERK1/2 phosphorylation was analyzed by using Western blotting with detection as described in Supporting Information S.2.4.4.