Duocarmycin A

Duocarmycin A (Duo), which is one of well-known antitumor antibiotics, efficiently alkylates adenine N3 at the 3' end of AT-rich sequences in the DNA. The addition of a minor groove binder, distamycin A (Dist), not only accerelates the reactivity of Duo with oligonucleotide duplex but also switches the DNA-alkylation site to guanine in GC-rich sequences. Here we examined cytotoxic effect of Duo in the coexistence of Dist using human lung carcinoma (HLC-2) cells. The cytotoxicity of Duo to HLC-2 cells was enhanced 10 times by the addition of 0.5microg/ml Dist, which was much lower than the IC(50) value of 16microg/ml. Addition of Duo alone to HLC-2 cells resulted in typically apoptotic changes, including chromatin condensation, sub-G1 accumulation in DNA histogram pattern, and decrease in procaspase-3 and 9 levels. Interestingly, these apoptotic characteristics in Duo-treated cells were suppressed by the addition of 0.5microg/ml Dist, and the G2/M population in the cell cycle progression of HLC-2 cells was largely unchanged in the coexistence of Dist along with the extremely low accumulation of p53 and higher induction of p21. In contrast, the treatment of HLC-2 cells with Dist (16microg/ml) alone was observed to induce the accumulation of p53 and cell cycle arrest at the G1 phase. These results indicate that Dist suppresses apoptosis induced by Duo as well as enhances Duo-induced cytotoxicity in living cells, and may contribute to chemotherapy for tumors resistant to inducing apoptotic cell death.

Duocarmycin A, a novel antitumor antibiotic, has a reactive cyclopropane ring, which has been reported to alkylate adenine at the 3' end of sequences of three or more consecutive A or T in DNA [Boger, D. L., et al. (1990) J. Am. Chem. Soc. 112, 8961-8971]. In order to study the DNA recognition, the reaction of DNA with duocarmycin A was performed in the presence of DNA ligands. Distamycin A, berenil, Hoechst 33258, and 4',6-diamidino-2-phenylindole (DAPI), which are minor-groove binders with affinity to A.T-rich sequences, were used. DNA-sequencing experiments showed that treatment of DNA with duocarmycin A plus distamycin A caused alkylation of guanine residues in G.C-rich sequences, which are not alkylated by duocarmycin A alone. Guanine alkylation by duocarmycin A was not observed with berenil, Hoechst 33258, or DAPI. HPLC product analysis showed that duocarmycin A reacted with a double-helical DNA octamer d(CCCCGGGG)2 in the presence of distamycin A to produce duocarmycin A-guanine adduct, while duocarmycin A alone did not react with the octamer. Chromomycin A3, which binds as a Mg(II)-coordinated dimer to G.C-rich sequences in the minor groove, inhibited the guanine alkylation by duocarmycin A in the presence of distamycin A. A footprinting experiment showed that there is a distamycin A-binding site close to the alkylated guanine residue. These results suggest that two different molecules, duocarmycin A and distamycin A, cooperatively recognize DNA sequences including consecutive G.C base pairs resulting in alkylation at the novel guanine sites. The cooperative drug recognition can be designated as "concerted DNA recognition".

A main problem of common cancer chemotherapy is the occurrence of severe side effects caused by insufficient selectivity of the applied drugs. A possible concept to overcome this limitation is light-driven prodrug monotherapy. The synthesis as well as photochemical and biological evaluation of new photoactivatable prodrugs is described. Best results were obtained with prodrug (S,S)-7a. The photochemical labile protecting groups in (S,S)-7a can easily be removed by irradiation with UV-A light in 30 min with a power of only 2 J cm(-2). The determination of the in vitro cytotoxicity by using an HTCFA-test reveals a QIC(50) value of 8200 and the prodrug is more than two million times less cytotoxic than the corresponding seco-drug (-)-(S,S)-5 with an IC(50) value of about 110 fM. The big therapeutic window makes (S,S)-7a very suitable for its use in selective cancer therapy.