Abstract: Cancer in most cases is still considered an incurable disease and thus there is a critical need to discover new anticancer drugs. In our efforts to search for novel anticancer agents or approaches, we have concentrated on three major groups of compounds, all of which act at least in part by inducing cancer cell death. Structure-function relationships of the newly synthesized compounds, selectivity, mode of action in killing cancer cells and cellular targets were explored.
The first group consists of compounds whose chemical structures comprises arylethylene moiety, the second group consists of iron chelators and the third group consists of naphthoquinones. Both known and newly synthesized compounds were studied for their antitumor efficacy in vitro on cell lines derived from hematological malignancies and some of the compounds were tested on cell lines derived from solid tumors. Based on structure-function relationship studies we found two active subgroups of compounds among the arylethylene derivatives namely, substituted 9-arylideneanthrones (BAs) and members of the triarylvinylic systems and two active groups among the naphthoquinones, namely, chloro- and pyrolidino-amino-phenyl-naphthoquinones. The iron chelators used are known compounds. The arylethylene derivatives exhibited a high degree of selectivity for cancer cells. Normal cells were relatively resistant to the naphthoquinones studied and iron chelators were active against different cell lines in a range of concentrations that are safe for human use.
The arylethylene derivatives and iron chelators killed the cells by apoptosis while the naphthoquinones induced apoptotic or necrotic cell death depending on time and concentration conditions. Apoptosis induced by members of the different groups was accompanied by the disruption of mitochondrial transmembrane potential and was followed by cytochrome c release and caspases activation.
Apoptosis induced by compounds from the three groups involves oxidative stress, which plays a critical role in mediating cell death. Nevertheless, the source of reactive oxygen species (ROS) varies. The different compounds were found to act through different signaling pathways. Using members of the triarylvinylic systems, the results suggest involvement of FLAP/5-LOX. However, the main sources for ROS production using BAs were NAD(P)H oxidase and mitochondria. The iron chelator mimosine caused formation of hydrogen peroxide and a decrease in reduced glutathione levels probably through its iron-chelating activity via catalase inhibition, while the results obtained with naphthoquinone derivatives suggest NAD(P)H-oxidase involvement in induction of oxidative stress and cell death.
The direct effect of the compounds on mitochondria was studied. The active naphthoquinones and mimosine induce mitochondrial swelling in isolated rat liver mitochondria, via opening of permeability transition pore. While the arylethylene derivatives tested from both subgroups, alone, do not cause mitochondria swelling. However, they protected mitochondria from Ca2+-induced swelling. It is possible that these compounds have a direct effect on the mitochondria causing stabilization of their membrane.
Some specific signaling steps were observed for the different compounds. PKC-? is specifically activated at early stage by the arylethylene derivatives. Our finding suggests a role for this enzyme in apoptosis mediated by members of the triarylvinyl family but not to those of the BA family. Treatment with the naphthoquinone derivatives induced a rapid phosphorylation of p38 mitogen-activated protein kinase (p38MAPK). Phosphorylation of extracellular signal-regulated protein kinases (ERK1/2) was observed as well. The results obtained imply that induction of p38 is involved in apoptosis mediated by naphthoquinones, whereas ERK1/2 plays role in cell survival and possibly in necrosis. ICL670 (ExjadeŽ, Deferasirox) a relatively new oral iron chelator was tested against mantle cell lymphoma cells. Our results indicate that the mechanism of ICL670 action includes shortening of cyclin D1 half-life via enhancement of protein degradation. The results point to the role of proteasome in this action.
The various groups of compounds affected the ratio of pro- and anti-apoptotic proteins of the Bcl-2 family. Differences in behaviour were obtained even within the same group of compounds. For example, an increase in the expression of Bax at both transcriptional and translational levels was seen with two active naphthoquinone derivatives. Moreover, most intriguing was the down regulation of Mcl-1 expression, which is one of the main anti-apoptotic proteins in leukemia, by one of the compounds while down regulation of Bcl-2, was seen with other compounds.
The effect of the different compounds was examined ex vivo on CLL and AML cells and on mononuclear cells obtained from healthy donors. The results indicated that the compounds killed the leukemic cells selectively and exhibited different specificities towards CLL and AML cells.
By virtue of the results obtained with members of the triarylvinylic systems, the Hematology Institute in Soroka University Medical Center began a phase I-II clinical trial to evaluate the potential therapeutic effect of clomiphene in advanced and refractory chronic lymphoblastic leukemia and acute myeloid leukemia patients. Our preliminary results revealed that the treatment caused stabilization of leukocyte levels during drug administration and slowed down disease progression in a few treated AML patients.
In summary, our results present novel and known compounds that act by the killing of cancer cells through various mechanisms and thus may provide effective anti-cancer strategies.