New approaches to the therapy of glioblastoma: investigations on RNA interference, kinesin Eg5 and ABCB1/ABCG2 inhibition

No effective chemotherapy for the treatment of malignant brain tumors, especially glioblastoma, exists so far. Despite the progress in surgical techniques and advances in the irradiation treatment, the concomitant chemotherapy is essential for the prevention of relapse and of major importance for patient outcome. The introduction of temozolomide combined with radiation in clinical practice led to slightly improved long-term survival, but malignant gliomas remain resistant to cancer chemotherapy. Thus, new strategies for the treatment of brain tumors are still needed. Objectives of this work were the evaluation of the TmHU protein as siRNA transfection reagent (Chapter 2), investigations on new kinesin Eg5 inhibitors, which specifically inhibit cell division during mitosis (Chapter 3), and the exploration of new tariquidar analogs as ABCB1 and ABCG2 inhibitors (Chapter 4). Due to the selective down-regulation of oncogene expression by small interfering RNA, siRNAs are considered as promising anticancer agents allowing the selective killing of tumor cells. The suitability of the non-toxic TmHU protein as siRNA transfection reagent was explored in vitro and in vivo in order to investigate the down-regulation of gene expression in human glioblastoma cells. The efficient transfection of the cancer cells is a prerequisite for this new concept, and, therefore, reliable in vitro and in vivo models were developed for the proof of principle. A suitable and convenient in vitro method was established for the quick detection of siRNA effects on the EGFP or DsRed2 expression, respectively, with respect to total cell number by using a fluorescence plate reader. In order to study siRNA effects in vivo subcutaneous tumor models were established in nude mice and two fluorescence detection methods were evaluated. The fluorescence intensity was quantified by confocal laser scanning microscopy of paraffin embedded tumor sections and by in vivo imaging. In preliminary tests with micro-osmotic pumps the continuous release of a suspension and the practicability of subcutaneous implantation were assured. Unfortunately, the established in vivo methods could not be used for the intended investigations on TmHU because this protein was not suitable for siRNA transfection. Nevertheless, these methods are recommendable for the investigation of promising in vivo transfection reagents. The inhibition of kinesin Eg5 by small molecules such as monastrol is currently evaluated as an approach to develop a novel class of antiproliferative drugs for the treatment of malignant tumors. Therefore, the effects of new monastrol analogs on the proliferation of human U-87 MG, U-118 MG, and U-373 MG glioblastoma cells were investigated. Compared to monastrol, the new cell cycle specific compounds showed an at least one order of magnitude higher antiproliferative activity. The compounds were neither inactivated by hydrolysis nor by binding to serum proteins. Due to the necessity of overcoming the blood�brain barrier (BBB) in the treatment of brain tumors, it was investigated if the new monastrol analogues are modulators or substrates of the ABCB1 transporter by a flow cytometric calcein-AM efflux assay. The tested compounds showed no modulating effects on the ABCB1 function. With respect to the treatment of primary and secondary CNS tumors, the results suggest that the new monastrol analogs represent an interesting class of potential anticancer drugs, predicted to be less neurotoxic in comparison to classical tubulin inhibitors. The efflux of cytostatics due to expression of ABC transporters such as ABCB1 and ABCG2 at the BBB leads to extremely low drug concentrations in the brain and is therefore a major limitation in cancer chemotherapy. A strategy to overcome the BBB is the administration of an efflux inhibitor in combination with a cytostatic. The 3rd generation inhibitor tariquidar led to better brain/plasma ratios of paclitaxel but could not increase the paclitaxel brain levels compared to co-administration of the 2nd generation inhibitor valspodar. The very high tariquidar brain levels indicate that the very lipophilic tariquidar is trapped in the lipid compartment of the brain. Aiming at tariquidar analogs with improved solubility and pharmacokinetics, more hydrophilic analogs were synthesized. The compounds were investigated on ABCB1 overexpressing Kb-V1 and ABCG2 overexpressing MCF-7/Topo cells for inhibitory activity and for cytotoxicity, alone and in combination with cytostatics. Surprisingly, slight structural modifications resulted in ABCG2 selective inhibitors. Three analogs have IC50 values of 55, 100, and 154 nM, comparable with the most potent reported ABCG2 inhibitor Ko143. Some ABCG2 inhibitors showed specific toxicity which could be advantageous regarding the treatment of brain tumors. These very potent and selective ABCG2 inhibitors should be useful pharmacological tools for in vitro and in vivo investigations.