A mechanistic assessment of novel anticancer drugs targeting the metastatic cascade and tumour vascularisation.

Angiogenesis, the process of induced blood vessel sprouting and vascularisation are essential for the growth and progression of solid tumours. Recruitment of blood vessels is also an important step of the metastatic cascade that enables development of macrometastasis and growth to secondary tumours. Thus, intratumoral blood vessels represent an important target for anticancer drugs. The two classes of antivascular chemotherapeutics are vascular-disrupting agents (VDA) that target already established tumour blood vessels, and compounds with antiangiogenic acitivity that interfere with processes during the formation of new blood vessels. Most vascular-disrupting agents bind to tubulin and mediate blood vessel destruction by secondary effects ensuing microtubule destabilisation. These include or are based on actin cytoskeleton reorganisation, cellular contractility and endothelial permeability. Eventually, the loss of the tightly-organised monolayer integrity of the endothelium results in blood vessel disruption and intratumoral haemorrhages. The tubulin-binding agents discussed in this thesis are derived from the synthetic quinazoline derivate Verubulin or the natural drug Combretastatin A-4 (CA-4). Their preclinical evaluation includes the determination of their in vitro cytotoxicity profile against a panel of different tumour cells lines and various biochemical and immunological methods to clarify their molecular and cellular mechanism of action. Both antimetastatic and antivascular activity of the best derivatives were assessed in vitro by using endothelial or tumour cell-based assays or in vivo by analyses of developing blood vessels within in the chorioallantois membrane (CAM) of chicken embryos. CA-4-derived oxazole and imidazole analogues that were synthesised and developed at the Chair of Organic Chemistry of the University of Bayreuth were able to overcome typical drawbacks of clinical CA-4 phosphate prodrugs including their short plasma half-life and metabolic instability as well as the induction of multidrug resistance (MDR)-mediating overexpression of efflux transporters. Unlike CA-4, imidazoles are chemically stable and do not undergo isomerisation into the inactive trans-configuration of the CA-4 stilbene motiv. Additionally, they are not recognised by MDR-transporters of cancer cell lines that are refractory to CA-4. In in vitro experiments, active derivatives led to extensive microtubule depolymerisation which we could correlate to their cytotoxicty and vascular-disrupting activity. The latter is a consequence of typical effects such as actin cytoskeleton remodelling and defective cellular adhesion dynamics. In addition, these drug-induced cytoskeletal alterations are also the origin of decreased tumour cell motility and reduced invasive behaviour. One of the most promising derivatives which is designated with its short name Brimamin, was shown to mediate perturbation of nuclear NF-kappaB signalling. Interference with this resistance mediator has not been described for CA-4 and is different from the mode of action of other antiangiogenic agents. A second generation of CA-4-derived imidazoles with acrylic hydroxamic acid appendages were shown to combine histone deacetylase (HDAC) inhibition with synergistic effects of the 4,5-diarylimidazole residue. Some of the derivatives proved a greater cytotoxicity against a panel of resistant cell lines and a higher specificity for cancer over non-malignant cells then the clinical approved HDAC inhibitor Vorinostat. The imidazoles also exceeded the antimetastatic in vitro acitivity and the antiangiogenic in vivo activity of Vorinostat. Like the latter, the best derivatives were shown to act as so called pan-HDAC inhibitors with unspecific inhibition of all Zn(II)-dependent HDAC isoenzymes, but a higer specificity for the tubulin deacetylase HDAC6. As a consequence, treatment with the best performing derivative with the short name Etacrox led not only to hyperacetylation of microtubules but also induced severe alterations in cytoskeletal and focal adhesion dynamics. An additional mode of action unique for the new imidazole-based HDAC inhibitors is the direct inhibition of matrix metalloproteinases (MMPs) which are known to promote angiogeneses and metastasis. The latter of pleiotropic effects together with HDAC inhibition-mediated perturbation of pro-angiogenic signalling cascades apparently mediates the strong antiangiogenic and antimetastatic effects. Altogether, the high tolerance of large doses in mice and their multitargeted anticancer effects make the 4,5-diarylimidazole HDAC inhibitors a promising class of new drug candidates for clinical applications.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Universität Bayreuth

Year

2015

Author dc:creator

• Mahal, Katharina

Contributors dc:contributor

• Schobert, Rainer

Repository record source_url

https://epub.uni-bayreuth.de/id/eprint/2062/

OAI identifier oai:identifier

oai:epub.uni-bayreuth.de:2062