Cinnamaldehyde and cuminaldehyde thiosemicarbazones and their copper(II) and nickel(II) complexes: A study to understand their biological activity Franco Bisceglie a,b , Silvana Pinelli b,c , Rossella Alinovi b,c , Matteo Goldoni c,d , Antonio Mutti c , Alessandro Camerini a , Lorenzo Piola a , Pieralberto Tarasconi a,b , Giorgio Pelosi a,b, ⁎ a Department of Chemistry, University of Parma, Parco Area delle Scienze 17/A, 43124 Parma, Italy b C.I.R.C.M.S.B. — Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi Biologici, Parma Local Unit, 43124 Parma, Italy c Department of Clinical and Experimental Medicine, University of Parma, Via Gramsci 14, 43126 Parma, Italy d Workers Compensation Authority (INAIL), Research Center at the University of Parma, Via Gramsci 14, 43126 Parma, Italy abstract article info Article history: Received 9 April 2014 Received in revised form 15 July 2014 Accepted 15 July 2014 Available online 23 July 2014 Keywords: Thiosemicarbazone Nickel Copper Caspase DNA Topoisomerase IIa This paper reports the synthesis and characterization of trans-cinnamaldehyde thiosemicarbazone (Htcin), cuminaldehyde thiosemicarbazone (Htcum) and their copper and nickel complexes. All the compounds, which on healthy cells (human fibroblasts) show a neglectable cytotoxicity, were screened in vitro in cell line U937 for their antileukemic activity. These compounds, in spite of their molecular similarity, present variegated behav- iors. Htcin shows no inhibition activity in U935 cells, while both its metal complexes inhibit proliferation with IC 50 at μM concentrations. The other ligand, Htcum, and its metal complexes, besides inhibiting proliferation, in- duce apoptosis. The cell cycle analysis highlights a G 2 /M checkpoint stop suggesting a possible direct action on DNA or on topoisomerase IIa. From CD and UV spectroscopy experiments, the DNA results to be not the main tar- get of all these molecules, while both copper complexes are effective topoisomerase IIa inhibitors. All of these molecules activate caspase-9 and caspase-3, while caspase-8 activity is significantly induced by both cinnamaldehyde metal complexes. Tests on PgP and intracellular metal concentrations (determined by mean of atomic absorption spectrometry) show that the compounds tend to accumulate in the cytoplasm and that the cells do not manage to pump out copper and nickel ions. © 2014 Elsevier Inc. All rights reserved. 1. Introduction Natural products have been a continuing source of novel drug leads [1] and, in this context, cinnamaldehyde has been tested for its antican- cer activity [2]. This molecule has recently shown very interesting results in cell cultures and animal models, but only at high doses not achievable through simple dietary intake [3]. Also cuminaldehyde has been under study to verify its effectiveness as an antitumor agent and has shown good results on cultured murine but not on cultured human cells [4]. On this basis, and with the aim to overcome the limits owned by the free aldehydes, we decided to modify their structure endowing them with donor atoms in order to render them potential ligands for metal ions. The derivatives we synthesized belong to the thiosemicarbazone fam- ily, which represents an interesting class of compounds (many of them being patented [5]) with a wide range of pharmacological applications thanks to their antibacterial, antiviral and antitumor activities [6–9]. In many cases, upon coordination to metal ions, the bioactivity of these com- pounds increases suggesting that complexation can be an interesting strategy of dose reduction [10–13]. Recently our studies addressed the synthesis of new SN chelating thiosemicarbazones derived from natural aldehydes such as citronellal, citral, octenal, retinal and their copper and nickel complexes [10,11,14–17]. We have then aimed our attention at the synthesis of new nickel and copper complexes of thiosemicarbazones derived from natural trans-cinnamaldehyde and cuminaldehyde (Htcin and Htcum). The corresponding thiosemicarbazones have also revealed antimicrobial and, more specifically, anti-tyrosinase activities [18,19] and many studies have been performed on DNA binding and nuclease activity of Cu(I) and Cu(II), and Pd(II) and Pt(II) complexes [20–22]. In the present work, after having synthesized and characterized cinnamaldehyde and cuminaldehyde thiosemicarbazones (Htcin 1 and Htcum 2) together with their Ni(II) and Cu(II) complexes ([Ni(tcin) 2 ] 3, [Ni(tcum) 2 ] 4, [Cu(tcin)Cl(OH 2 )] 5 and [Cu(tcum)Cl(OH 2 )] 6), a series of biological assays were performed. We started from a comparison between their proliferation inhibition on human histiocytic lymphoma cell line U937 vs human fibroblasts (non-cancer cells) and, subsequently, we carried out a series of more specific tests such as metal accumulation in the cytoplasm, Pgp protein activation, cell cycle assay, caspase-3, -8, and -9 assays, reactive oxygen species (ROS) determination, DNA interac- tion and topoisomerase IIa inhibition. Journal of Inorganic Biochemistry 140 (2014) 111–125 ⁎ Corresponding author. Fax: +39 0521905420. E-mail address: giorgio.pelosi@unipr.it (G. Pelosi). http://dx.doi.org/10.1016/j.jinorgbio.2014.07.014 0162-0134/© 2014 Elsevier Inc. All rights reserved. Contents lists available at ScienceDirect Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio