A Pt(IV) Prodrug Combining Chlorambucil and Cisplatin: a Dual-Acting Weapon for Targeting DNA in Cancer Cells

Abstract

In this study, two DNA-targeting agents, cisplatin and chlorambucil, were combined in a Pt(IV) prodrug, 1, which was thoroughly characterized by means of spectroscopic and spectrometric techniques. Tested towards a panel of various human tumor cell lines, this compound showed superior in vitro antitumor potential than the reference drug cisplatin. In addition, an antitumor potential of 1 was found, which is comparable to that of oxaliplatin in 3D spheroid models of colon cancer cells. Mechanistic studies performed in colon cancer cells confirmed that the conjugation of chlorambucil to Pt(IV) cisplatin-based scaffold tunes the lipophilicity of the prodrug, consequently improving the ability of the compound to accumulate into cancer cells and to target DNA, ultimately leading to apoptotic cancer cell death.

Keywords: Pt(IV) prodrugs; chlorambucil; cisplatin; cytotoxicity.

Scheme 1

Structure of cisplatin, chlorambucil, and of 1.

Scheme 2

Synthetic pathway for 1.

Figure 1

HPLC chromatograms of complex 1 in DMSO/PBS (1/10) at 37 °C.

Figure 2

HPLC chromatograms of the reduction of complex 1 by ascorbic acid (AA). (* is AA) (See Materials and Methods for details).

Figure 3

Cellular uptake (A) and DNA platination (B). (A) HCT-15 cells were incubated for 24 h with increasing concentrations of 1 or CDDP. The error bars indicate SD. (B) Platination levels of nuclear DNA extracts. HCT-15 cells were treated for 24 h with increasing concentrations of 1 or CDDP. DNA was extracted, quantified, and the amount of Pt bound to DNA was estimated using GF–AAS. Error bars indicate SD.

Figure 4

DNA fragmentation and apoptosis induction. (A) Nuclear DNA fragmentation. HCT-15 cells were treated for 24 or 48 h with IC₅₀ of 1 or CDDP. Quantitative estimation of DNA fragmentation was obtained with an ELISA test. Error bars indicate SD. ** p < 0.01. (B) HCT-15 cells were treated with IC₅₀ of 1 or CDDP for 48 h and stained with the fluorescent dye Hoechst 33258. (a) control cells; (b) CDDP-treated cells; (c) 1-treated cells.