Abstract C101: Subcellular distribution study by means of the inherent fluorescence of the investigational drug Triapine and its zinc(II) complex

Abstract

Abstract The antineoplastic activity of α-N-heterocyclic thiosemicarbazones was discovered several decades ago. Currently the most promising drug candidate of this class of compounds is Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone, 3-AP), which entered several phase I and II clinical trials as an antitumor agent. We discovered that Triapine possesses intrinsic fluorescence properties. This enabled us to monitor for the first time the uptake and intracellular distribution of an α-N-heterocyclic thiosemicarbazone in living human cancer cells by fluorescence microscopy. In addition we synthesized the first zinc(II) complex [Zn(Triapine)Cl2]•HCl of Triapine, which shows fluorescence as well, compared its cytotoxicity in human cancer cells with that of the parental compound and studied the influence of metal complexation on intracellular distribution. Triapine and its zinc complex were characterized spectroscopically. The UV-Vis spectrum of Triapine in water shows a maximum absorbtion band at 359 nm, whereas the maximum absorbtion of [Zn(Triapine)Cl2]•HCl is slightly shifted to 365 nm. The maxima of the emission spectra of both compounds are at 457 nm when irradiated at ex = 360 nm. Fluorescence microscopy of living SW480 (colon carcinoma) cells treated with Triapine or [Zn(Triapine)Cl2]•HCl showed that both compounds have a striking affinity to the nuclear membrane and to an organelle structure in the cytoplasm, whereas the localization in the nucleus was strongly different. The zinc complex binds to substructures within the nucleus, which could be verified as nucleoli by immunostaining of the nucleolar protein fibrillarin and co-staining with [Zn(Triapine)Cl2]•HCl. On the contrary, a strong affinity of Triapine to the nucleoli was not detected. The cytotoxic potencies of Triapine and [Zn(Triapine)Cl2]•HCl were determined in SW480 (colon carcinoma) and 41M (ovarian carcinoma) cells by means of the colorimetric MTT assay. The IC50 value of Triapine is 0.55±0.2 µM in SW480 cells and 0.45±0.03 µM in the 41M cell line. [Zn(Triapine)Cl2]•HCl showed IC50 values of 0.54±0.02 µM in SW480 and 0.52±0.02 µM in 41M cells. Comparing the two substances, no remarkable differences were observed. Our results demonstrate that Triapine can be visualized with fluorescence microscopy in living cells without any conjugation of fluorophores. Furthermore our findings indicate that metal complexation leads to a different subcellular distribution. Additional studies are ongoing to clearify the influence of metal complexation on the mode of action. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C101.