Over the last decades extensive research has shown that ROS and antioxidant defence systems play an important role in the process of tumour development and progression. Therefore, inhibition of members of the thioredoxin family may contribute to successful cancer therapy. Numerous effective natural and synthetic Txnrd inhibitors are described to possess anti-tumour potential. The mode of action ranges from induction of oxidative stress to cell cycle arrest and apoptosis. Most of these drugs target the Sec-containing active site of Txnrds, e.g. gold compounds, platinum compounds, arsenic trioxide, motexafin gadolinium, nitrous compounds and various flavonoids, extensively summarised by Urig and Tonissen 310,
316
. Several studies reported on gold(III)-compounds that specifically inhibit Txnrd2, thereby leading to Ca2+-dependent mitochondrial membrane permeability followed by cytochrom C release and induction of apoptosis 64, 268-269. Motexafin gadolinium (MGd), a drug that was reported to undergo redox cycling and to generate superoxide and other ROS, has been also shown to inhibit Txnrds and ribonucleotide reductase 78-79, 132, 193-194, 266. Currently MGd was tested successfully already in clinical trials (phase I-III) as single drug or in combination with other chemotherapeutic agents and/or radiotherapy for the treatment of different types of cancer 4, 80, 207, 314.
Also BSO, a selective inhibitor of intracellular GSH-synthesis, has been shown to effectively enhance the cytotoxicity of cisplatin-resistant tumours and the anti-tumour activity of the alkylating agent melphalan 55, 277. Previous work in our laboratory demonstrated, targeting more than one redox-regulating system might be a promising approach for anti-cancer therapy 197.
The outcome of the present study suggests that inhibition of Txnrd2 alone might offer an efficient way to interfere with cancer growth. Depletion of the GSH-dependent system in tumour-bearing mice seems to provide an additional benefit in reducing tumour growth. The
in vitro data of the present work only partially indicate an impact of Txnrd2 on tumour cell proliferation and colonigenic potential, whereas our in vivo data show that genetic deletion of
Txnrd2 affects tumour progression, most likely due to impaired vessel recruitment and
tumour vascularisation. However, further research is needed to fully clarify the role of Txnrd2 in proliferation, angiogenesis and endothelial cell function, especially in context of the discrepancies between in vitro and in vivo findings. Until now most research has been performed to investigate the cellular functions of the cytosolic counterpart in the context of tumour angiogenesis and endothelial cell function 72, 162, 219, 297, whereas only some studies
implicate the mitochondrial thioredoxin-dependent system in endothelial function 65, 68, 212, 354. To further explore the role of Txnrd2 in endothelial function and tumour growth, we
established the tamoxifen-inducible endothelial-specific Txnrd2-knockout mice (chapter 3.4.2). The endothelial-specific Txnrd2-knockout mice are viable and thus will offer an efficient tool to further investigate the role of Txnrd2 in endothelial function and tumour angiogenesis in vivo. To study the signalling between tumour cells and endothelial cells we are currently also establishing an in vitro endothelial sprouting assay 227, 229, 278. This model will allow us to study the consequences of genetic deletion of Txnrd2 in endothelial cells and/ or tumour cells in the process of endothelial cell sprouting.
5.
SUMMARY
The mitochondria specific thioredoxin-dependent system consists of thioredoxin 2 (Trx2), thioredoxin reductase 2 (Txnrd2) and thioredoxin-dependent peroxidases (Prx3 and Prx5). Along with the glutathione (GSH)-dependent system it is critically involved in the maintenance of an intracellular redox balance.
Previous studies revealed that primary Txnrd2-deficient mouse embryonic fibroblasts (MEFs) show impaired proliferation, produce increased levels of reactive oxygen species (ROS) and are highly susceptible towards several pro-oxidants as well as depletion of the intracellular GSH.
In contrast, Txnrd2-null MEFs which continuously lack Txnrd2 seemed to compensate for Txnrd2-deficiency by upregulation of other redox-regulating systems. Additionally, these cells switched their energy metabolism towards anaerobic glycolysis in favour to oxidative phosphorylation to protect themselves from a potentially increased formation of mitochondrial ROS.
The main objective of the current study was to analyse the impact of Txnrd2 on tumour growth. Indeed we could show that loss of Txnrd2 strongly impairs the colonigenic potential of tumour cells whereas proliferation and ROS level were unaffected. Transformed Txnrd2- null cells were highly susceptible to depletion of intracellular GSH.
In vivo studies revealed that deletion of Txnrd2 resulted in 50% reduction in tumour size which was accompanied by reduced proliferation due to impaired formation of tumour vessels. These phenomena could be attributed to reduced Hif-1α and VEGF protein expression. In agreement with the in vitro data additional therapeutic treatment of mice bearing Txnrd2-null tumours with L-buthionine sulfoximine (BSO), revealed increased susceptibility of the Txnrd2-null tumours towards GSH-depletion and resulted in further reduction in tumour size about 38%. Altogether, our results identify Txnrd2 as a promising drug target for cancer therapy. Furthermore the dual inhibition of Txnrd2 and GSH- dependent system, offers an attractive strategy to combat tumour growth.
The second part of the study investigated, whether Txnrd2 could influence endothelial cell proliferation and angiogenic function directly. Therefore, wild-type and Txnrd2-deficient embryonic endothelial progenitor cells (eEPCs) were isolated and cultivated. In vitro
proliferation of Txnrd2-null eEPCs was only slightly diminished. In tube formation assays, the cells showed impaired angiogenic capacity, indicating that Txnrd2 might be indeed pivotal for endothelial proangiogenic function.