Cytotoxic ribosome-inactivating proteins (RIPs) of type II such as ricin were investigated as anti-cancer agents, but also pose a threat as biological weapons. The molecular mechanism leading to their toxic effects is, however, not yet clear. The current paradigm, which states that the irreversible depurination of 28S rRNA results in a general translational arrest eventually leading to cell death, has been questioned. Using micro-array, qRT-PCR and Western blot, we identified the unfolded protein response (UPR), a cellular mechanism activated in response to endoplasmic reticulum stress, that is induced in HCT116 and MDA-MB-231 cells exposed to the plant type II RIPs ricin, riproximin and volkensin. Apoptosis was induced by concentrations at which translation of UPR-related genes still occurred, despite concomitant ribosomal depurination. We conclude that UPR induction represents a model that better describes the cellular effects of RIP exposure at concentrations at which selected proteins are translated despite ribosomal depurination.

BACKGROUND AND PURPOSEMethyl jasmonate (MJ) is a plant stress hormone with selective cytotoxic anti-cancer activities. The TNF-related apoptosis-inducing ligand (TRAIL) death pathway is an attractive target for cancer therapy. Although TRAIL receptors are specifically expressed in primary cancer cells and cancer cell lines, many types of cancer cells remain resistant to TRAILinduced cytotoxicity. Here we have assessed a possible synergy between MJ and TRAIL cytotoxicity in colorectal cancer (CRC) cell lines. EXPERIMENTAL APPROACHCRC cell lines were pre-incubated with sub-cytotoxic concentrations of MJ followed by TRAIL administration. Cell death was determined by XTT assay and microscopy. Cytochrome c release, caspase cleavage, TRAIL-associated factors, X-linked inhibitor of apoptosis (XIAP) and survivin protein levels were detected by immunoblotting. Survivin transcription was examined by RT-PCR. KEY RESULTSPre-treatment with MJ resulted in increased TRAIL-induced apoptotic cell death, increased cytochrome c release and caspase cleavage. TNFRSF10A, TNFRSF10B, TNFRSF10D, Fas-associated death domain and cellular FLICE-like inhibitory protein remained unchanged during MJ-induced TRAIL sensitization, whereas MJ induced a significant decrease in survivin protein levels. Overexpression of survivin prevented MJ-induced TRAIL cytotoxicity, implying a role for survivin in MJ-induced TRAIL sensitization. MJ decreased survivin mRNA indicating that MJ may affect survivin transcription. In a b-catenin/transcription factor (TCF)-dependent luciferase activity assay, MJ decreased TCF-dependent transcriptional activity. CONCLUSION AND IMPLICATIONSMJ, at sub-cytotoxic levels, sensitized CRC cells to TRAIL-induced apoptosis. Thus, combinations of MJ and TRAIL, both selective anti-cancer agents, have potential as novel treatments for CRC. AbbreviationscFLIP, cellular FLICE-like inhibitory protein; CRC, colorectal cancer; FADD, Fas-associated death domain; IAP, inhibitors of apoptosis; MJ, methyl jasmonate; PARP, poly ADP ribose polymerase; TRAIL, TNF-related apoptosis-inducing ligand