A potent cytotoxic photoactivated platinum complex

Abstract

We show by x-ray crystallography that the complex trans, trans, trans-[Pt(N(3))(2)(OH)(2)(NH(3))(py)] (1) contains an octahedral Pt(IV) center with almost linear azido ligands. Complex 1 is remarkably stable in the dark, even in the presence of cellular reducing agents such as glutathione, but readily undergoes photoinduced ligand substitution and photoreduction reactions. When 1 is photoactivated in cells, it is highly toxic: 13-80 x more cytotoxic than the Pt(II) anticancer drug cisplatin, and ca. 15 x more cytotoxic toward cisplatin-resistant human ovarian cancer cells. Cisplatin targets DNA, and DNA platination levels induced in HaCaT skin cells by 1 were similar to those of cisplatin. However, cisplatin forms mainly intrastrand cis diguanine cross-links on DNA between neighboring nucleotides, whereas photoactivated complex 1 rapidly forms unusual trans azido/guanine, and then trans diguanine Pt(II) adducts, which are probably mainly intrastrand cross-links between two guanines separated by a third base. DNA interstrand and DNA-protein cross-links were also detected. Importantly, DNA repair synthesis on plasmid DNA platinated by photoactivated 1 was markedly lower than for cisplatin or its isomer transplatin (an inactive complex). Single-cell electrophoresis experiments also demonstrated that the DNA damage is different from that induced by cisplatin or transplatin. Cell death is not solely dependent on activation of the caspase 3 pathway, and, in contrast to cisplatin, p53 protein did not accumulate in cells after photosensitization of 1. The trans diazido Pt(IV) complex 1 therefore has remarkable properties and is a candidate for use in photoactivated cancer chemotherapy.

Fig. 1.

X-ray crystal structure of 1. Selected bond lengths (Å) and angles (°): Pt-N(1) 2.059(4); Pt-N(2) 2.020(4); Pt-N(3) 2.044(3); Pt-N(21) 2.046(3); N(11)–N(12) 1.203(5); N(12)–N(13) 1.139(6); N(21)–N(22) 1.216(5); N(22)–N(23) 1.138(5); N(11)–N(12)–N(13) 174.4(5); N(21)–N(22)–N(23) 175.9(5).

Fig. 2.

Photoreactions of 1. (A) UV-visible spectra recorded after 0, 1, 5, 15, 30, and 60 min of UVA irradiation. (B) Two-dimensional [¹H,¹⁵N] HSQC NMR spectra for ¹⁵N-1 and 2 mol equiv 5′-GMP after 1 and 30 min of irradiation. *, ¹⁹⁵Pt satellites. (C) Decrease in concentration of 1 and formation of 4 and 5 with increasing irradiation time.

Fig. 3.

Phototoxicity of complex 1. Human HaCaT keratinocytes (A), A2780 human ovarian carcinoma cells (B), and A2780cis cisplatin-resistant cells (C) were exposed to 5 J/cm² filtered UVA or sham irradiated. (D) HaCaT keratinocytes exposed to 5 J/cm² filtered TL03 visible light (λ_max 420 nm). The viability of cells treated with TL03 radiation alone was 111.4%. Open symbols, sham–irradiated samples; filled symbols, irradiated samples; circles, complex 1; squares, cisplatin.

Fig. 4.

In vitro repair synthesis assay of the extract prepared from the repair-proficient HeLa cell line. Repair synthesis used as substrates nonmodified pBR322 plasmid and pUC19 plasmid nonmodifed (lane noPt) or modified at r_b = 0.05 by cisplatin or transplatin in the dark (lanes cisPt and transPt, respectively), or by complex 1 under irradiation conditions (lane 1). (A) Results of typical experiment. (Upper) Photograph of the ethidium-stained gel. (Lower) Autoradiogram of the gel showing incorporation of [α-³²P]dCMP. (B) Incorporation of dCMP into plasmid modified by cisplatin, transplatin or complex 1. For all quantifications, representing mean values of three separate experiments, incorporation of radioactive material was corrected for the relative DNA content in each band. Radioactivity associated with incorporation of [α-³²P]dCMP into DNA modified by cisplatin was taken as 100%.