Thiosemicarbazones as anticancer agents

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121 but altered DNA conformation creating knot-like structures and hairpins (Buschini et al., 2014).

We have hypothesized previously that the DNA damage induced by the nickel complex could be due to a direct interaction between the complex and the DNA backbone and/or histones, giving rise to structural alterations of chromatin, such as heterochromatinization, that could interfere with a correct mitosis processes inducing apoptosis. To understand if the results obtained from the Alkaline Comet Assay, showing an important DNA migration starting from 1h treatment, could be related to a real DNA damage or to an altered DNA conformation that could produce alkali labile sites, we analysed the transcription profile of genes involved in DDR (such as ATM, ATR, Chk1, Chk2, Cyclin A1 and B). Identifying molecular pathways targeted by a compound is of paramount importance for the development of new drugs and the prediction of its mechanism of action has been attempted by using transcriptional expression profiles following drug treatment (Iorio et al., 2010).

Under genotoxic stress, the activation of ATM and/or ATR, DNA sensors for the initial response to single and double stranded DNA breaks, is a result of the formation their monomers or the induction of their transcription. After the treatment with the nickel complex, we did not observe an activation of the transcription of ATM and ATR. These results corroborate our hypothesis that [Ni(tcitr)2] did not induce SSB and/or DSB breaks, directly or indirectly. This data is in agreement with the lack of in vitro clastogen activity of [Ni(tcitr)2] on supercoiled DNA plasmid pBR322, previously reported (Buschini et al., 2014).

We did not observe alterations in the mRNA levels of ATM and ATR, but in contrast we found a strong up-regulation of the DNA damage sensors, Chk1 and Chk2. Probably, the metal complex could activate alternative pathways that trigger the transcription of Chk1 and Chk2.

[Ni(tcitr)2] caused also an important up-regulation of Cyclin B, that together with Cdk1, is involved in the cell cycle progression. In stress condition, Cyclin B could interact with APC (anaphase promoting complex), which plays a central role in regulating mitosis and the G1 phase of the cell cycle. Furthermore, APC induces the degradation of cyclin B and inhibits cell cycle progression. This up-regulation could lead to the cell cycle block and could interfere with the normal transition from G2 phase to M phase, as reported for [Ni(tcitr)2].

122 These results could support a strong interference of [Ni(tcitr)2] with the correct folding of chromosome during mitosis leading to apoptosis. Further analysis on proteins involved in the mitotic checkpoint could be important to better understand the relationship between DNA- [Ni(tcitr)2] interactions and cellular toxicity.

Several thiosemicarbazones inhibit the small subunit of the ribonucleotide reductase, that represents probably the best described target of thiosemicarbazones. This metalloenzyme is crucial for DNA synthesis as well as DNA damage repair and is frequently overexpressed in cancer cells making it an attractive target for the treatment. Inhibition of the tyrosyl radical in the active center of the RRM2/p53R2 subunit was demonstrated for several thiosemicarbazones (including Triapine and Dp44mT). Furthermore, previous studies in Saccharomyces cerevisiae to clarify the action mechanism of [Ni(tcitr)2] have shown, after analysis of collection of deletants, an enrichment in the classes of genes coding for components involved in nucleic acids metabolism such as ribonucleotide reductase (RR).

To identify another possible target of the metal complex, U937 cells were treated with a toxic concentration and a subtoxic one of each compound. The nickel complex induced a very important modulation of the different subunits of RR. Probably, the strong RRM2 up-regulation could indicate that the compound could act targeting specifically this subunit of the enzyme. We analysed also the expression of p53R2 in order to determine if the cellular response induced by the metal complex could involve p53. We did not observe alteration in the transcription level of the p53R2 subunit: this result highlighted that the [Ni(tcitr)2] could act with a p53-independent mechanism, as hypothesized before (Buschini et al., 2009).

We used the same approach to identify the molecular action mechanisms of the copper and the platinum complexes. Both induced a significant DNA damage highlighted through the Alkaline Comet Assay. DNA migration induced after 1h by [Cu(tcitr)2] or [Pt(tcitr)2] was less than the one induced by [Ni(tcitr)2]. We presume that the DNA damage observed after the treatment with [Cu(tcitr)2] could be due to an excessive production of ROS species, due to the fact that Cu could present different oxidation states in the cell. Oxidative stress due to ROS is known to cause DNA lesions of both SSB and DSB nature through the direct interaction of ROS with DNA (Rahal et al., 2016). We did not observe an activation of the

123 transcriptional profile of ATM and ATR but we showed an up-regulation of Chk1.

Furthermore, [Cu(tcitr)2] was not able to induce a cell cycle arrest (Bisceglie et al., 2012).

In HNC cells, the anticancer activity of [Cu(tcitr)2] would not seem to be related to p53 mutational status (wt or mutant). Also in other studies regarding DpT analogs, p53 status did not affect the anticancer effect, indicating a p53-independent mechanism of antiproliferative activity (Heffeter et al., 2018). [Cu(tcitr)2] treatment induced a WT-like expression in the p53 protein that restores p53 status: we hypothesize that this compound could restore the wild-type function of the p53 protein. This mechanism of action was proposed also for other thiosemicarbazones, such as COTI-2 (Heffeter et al., 2018).

It could be speculated that the down-regulation of both the ATM-ATR kinases, as we reported, could explain the decrease of p53 protein expression. p53 is regulated through phosphorylation of p53 protein at serine 15, mediated by ATM or ATR, to enhance its transactivating activity. Several studies highlighted that the down-regulation of ATM and ATR induced the degradation of p53 by MDM2 (Yang et al., 2004).

The platinum complex induced a strong DNA damage and acts as a promutagen agent. Most of the platinum anticancer agents target DNA. The connections between cell cycle and cell death were studied: we investigated if the treatment with the metal complexes could involve an alteration in the progression of cell cycle. Differently from [Ni(tcitr)2], [Pt(tcitr)2] induced a block in S phase as for cisplatin in several cancer cell lines (Mueller et al., 2006).

Also for this molecule, no transcription of ATM and ATR was detected, while Chk2 was upregulated after 24h treatment. Chk2 is directly involved in G1-S cell cycle arrest. Cisplatin usually activates DDR through ATM involvement. It will be interesting to understand how [Pt(tcitr)2] is able to modulate the cell cycle progression without modulating ATM transcription. It will be necessary to study the phosphorylation state of ATM protein after [Pt(tcitr)2] treatment.

Methylation strategy plays an important role in drug design: a lot of studies highlighted the importance of methyl groups in modulating biological activity, selectivity, solubility, metabolism and pharmacokinetic/pharmacodynamic properties of biologically active molecules. The significant improvements in activity are usually the result of the methyl group’s capacity to fill a hydrophobic pocket of the protein and/or to induce a conformational change of the molecule. Furthermore, addition of a methyl group to a specific molecule usually leads to an increase in lipophilicity and therefore a decrease in

124 water solubility (Sun et al., 2018). Starting from platinum, copper and nickel complexes, we measured the antiproliferative activity of dimethylated molecules against U937 cells. These dimethylated complexes displayed a mild anticancer effect compared to the parental compounds. The introduction of a methyl group in the skeleton of the starting molecules seems to be related to a decrease in their antitumor effects.

As for the parent molecules, the dimethylated complexes interacted with DNA and caused a DNA damage. The results indicate that the dimethylate compounds are less genotoxic than the starting molecules.

This study highlighted that the biological activity of our compounds depends on the metal ion used in the coordination process. Metals play a key role in the anticancer activity of thiosemicarbazones and the corresponding metal complexes represent an emerging class of experimental anticancer chemotherapeutic agents which shows in vitro antiproliferative activities.

The metal complexes could act as multitarget agents. The first target could be the DNA molecule and the second one could be the ribonucleotide reductase enzyme. Finally, the metal complexes are able to trigger several cellular pathways involved in DNA damage response and cell cycle progression.

In conclusion, these experimental data confirm that thiosemicarbazone scaffold represents a good starting point for the development of new anticancer agents.