35 The end goal of this project is the study of the biological activity of newly synthesized metal complexes, deriving from natural products, in order to identify their cellular targets and molecular action mechanisms. The exploitation of bioactive natural sources to obtain new agents with novel modes of action may represent an innovative and successful strategy in the field of medicinal chemistry. Many natural products and their chemical analogues have been proposed as starting molecules to synthetize compounds with increased biological potential. The tested molecules belong to the class of thiosemicarbazones, very attractive metal-chelating ligands, that show coordinating versatility and the possibility to easily modify the molecular backbone, tuning their physical and chemical properties.
This thesis can be divided into three sections.
▪ Aim 1.
The first section is part of ”Aflatox project“ focused on the design and the synthesis of new types of inhibitors of proliferation and aflatoxins production of Aspergillus flavus. In this context, we developed a new approach to assess toxicity and genotoxicity of the new compounds in order to identify their harmless effects to the environment and to the human health. All these results have contributed to the creation of a database correlating chemical structures and biological/toxicological activities.
▪ Aim 2.
In this section, we investigated the antiproliferative potential of different thiosemicarbazone metal complexes.
In our laboratory, citronellal, vanillin, morpholin, cinnamaldehyde and cuminaldehyde have been chosen as starting aldehydes to prepare different thiosemicarbazones and their corresponding metal complexes with the aim to study how substituent groups could influence their biological activity.
In previous studies, the free ligands did not show any significant cytotoxic effect, independently from the presence or the absence of a substituent on the terminal aminic nitrogen. On the contrary, metal complexes showed marked biological activity: for example, copper complexes with the morpholine ligand presented a highest inhibition of proliferation on central nervous system cell line SN-K-MC, cervical cancer cells HeLa and leukemia cells Jurkat and K562 (Bisceglie et al., 2018). Furthermore, the nickel(II) and the
36 copper(II) complexes of cinnamaldehyde and cuminaldehyde thiosemicarbazones showed antileukemic activity on U937 human cell line. Metal complexes of cuminaldehyde thiosemicarbazone cause G2/M phase cell cycle arrest suggesting a possible action on topoisomerase II. All of these molecules activate caspase-9 and caspase-3, while caspase-8 activity is significantly induced by both cinnamaldehyde metal complexes (Bisceglie et al., 2014).
In other studies, citronellal was used to synthetize the nickel ([Ni(tcitr)2]) (Buschini et al., 2009) and the copper ([Cu(tcitr)2]) (Bisceglie et al., 2012) complexes, where tcitr = citronellalthiosemicarbazonate. Both the complexes demonstrated a strong cytotoxic activity against a panel of cancer cells. In particular, many analyses were carried out employing a leukemia cell line (U937). [Ni(tcitr)2]is not active on G0 cells (fresh leukocytes) but is able to induce perturbation of the cell cycle on stimulated lymphocytes and U937 cells, in which a G2/M block was detected. The nickel complex causes p53 independent-intrinsic-apoptosis via down-regulation of Bcl-2, mitochondrial membrane potential loss, caspases-3 and -9 activation (Buschini et al., 2009). [Ni(tcitr)2] also caused DNA damage but neither mutagenicity nor recovery were detected. It interacted with DNA and altered DNA conformation creating knot-like structures and hairpins but it did not induce gene mutation or chromosomal damage (Buschini et al., 2014). The action mechanism is still unclear but this nickel complex is a promising candidate for the synthesis of new metal thiosemicarbazones with potential biological activity.
In this research, all these experimental data were used to better understand the action mechanism of [Ni(tcitr)2], [Cu(tcitr)2] and a new platinum complex [Pt(tcitr)2]. Starting from these metal complexes, we detected the antiproliferative activity of their corresponding dimethylated derivatives. All compounds were screened for their cytotoxic effect on a panel of human cancer cell lines selected from the National Cancer Institute “60 Human Tumor Cell Line Anticancer Drug Screen”. We performed also in vitro studies to detect genotoxic and mutagenic activities and to identity an interaction of metal complexes with DNA.
Human leukemic U937 cell line was used as an in vitro model for mRNA expression studies.
First, we examined the modulation of the transcription of the enzyme ribonucleotide
37 reductase (RR) as possible target of the metal complexes. Indeed, many thiosemicarbazones target RR, interfering with the essential di-iron tyrosyl radical center of its small subunit.
Subsequently, DNA damage induced by metal complexes treatment has been related to mRNA levels of several proteins that regulate DNA damage response and genome integrity.
In particular, we focused on the DNA damage sensors, Chk1 and Chk2, that participate in G2/M checkpoint control through the ataxia telangiectasia mutated (ATM)/ATM RAD3 related (ATR) pathway. To determine the correlation between proliferation inhibition and cell cycle blockage, we analysed the expression levels of cyclin A/Cdk2 complex, that are known to participate in the initiation of mitosis in human cancer cells, and of cyclin B, a key component involved in G2 to M phase transition. Furthermore, in order to understand metal complexes transport into U937 cells, we studied hCTR1, a transmembrane protein that is involved in the uptake of platinum anticancer drugs.
▪ Aim 3
The last section of this project was carried out at the International Agency for Research on Cancer (IARC). Cytotoxic activity of metal complexes was investigated using different cell lines resulting from primary tumour samples and collected as part of a multicenter case-control study coordinated by IARC. Cells were selected for a different pattern of TP53 mutations. p53 is a tumor suppressor protein and its mutation leading to loss of wild-type p53 activity has been frequently detected in multiple cancer types. Furthermore, perturbations in p53 signaling pathways were required to cancer development and progression. Cells sensitivity was correlated with TP53 status in order to understand if the cytotoxic mechanisms of the compounds could occur through a p53-dependent pathway.
In this study, the copper complex exhibits the strong antiproliferative activity against tumour samples.
Subsequently, through western blot analysis we determined different post translational modifications of p53 protein induced by the treatment with copper complex. We studied several p53 post-translational modifications, such as acetylation and phosphorylation in specific sites, induced when cells were exposed to genotoxic stimuli.
38 In conclusion, these results highlight that the treatment with metal complexes could trigger specific cellular pathways leading to apoptosis. Furthermore, thiosemicarbazone scaffold represents a good starting point for the development of new anticancer agents.