Table of Contents

Israeli Principal Investigator

Prof. Eliezer Flescher
Department of Human Microbiology
Sackler Faculty of Medicine
Tel Aviv University, Tel Aviv, Israel

DKFZ Principal Investigator

Prof. Martin R. Berger
Toxicology and Chemotherapy Unit
German Cancer Research Center, Heidelberg, Germany

Approaches and Achievements

The drawing shows the molecular model of riproximin and depicts the catalytic site of the A chain (red), the intermolecular disulfide bridge (yellow), the B chain sugar binding domains 1 (green) and 2 (pink) as well as the potential N-glycosylation sites (dark blue). | © dkfz.de

Riproximin (rpx), a ribosome-inactivating protein (RIP) of type II possesses potent anticancer activity in vitro and in vivo (Voss et al., 2006). RIPs of type II affect and damage cells by different mechanisms.
We performed micro-array studies to analyse the mode of action of riproximin at the level of gene transcription. The analysis of the chip results revealed that riproximin caused an ER stress response in breast adenocarcinoma MDA-MB-231 cells. ER stress related genes like the transcription factor ATF3 were significantly up regulated after treatment with this agent, even at lower concentrations.
These RNA based data were further confirmed at protein level. By Western blot prominent ER stress markers like the phosphorylated form of eIF2Ą and increased ATF3 levels could be detected within 8 hours. Furthermore the assumption that both, an excessive RIP load or a long exposure will lead to apoptotic cell death could be confirmed by the presence of cleaved PARP, which in its intact form is an effector caspase substrate in apoptosis. An additional FACS experiment further supported this model. The Western blot studies were extended to other RIPs (volkensin, ricin) and cell lines (HCT116, HeLa). The results were comparable to those of riproximin in MDA-MB-231 cells.

MDA-MB-231 cells were grown in cell culture plates for 24 h and then treated for additional 24 h with various concentrations of riproximin (4-500 pM). The cells were harvested and lysed in RIPA buffer. Equal protein amounts were separated by SDS-PAGE. The Western blot analysis was carried out with primary antibodies against ATF3 and ERK-2 (loading control). | © dkfz.de

We discovered a new mode of action of ribosome-inactivating proteins in plants which finally leads to cell death. Via an ER stress response cancer cells first react with cell cycle arrest until they finally undergo apoptosis when the exposure to the drug becomes too high.