Verrucarin A

We investigated whether verrucarin A (VA) sensitizes HepG2 hepatoma cells to tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis. We found that VA alone induces little apoptosis, but when combined with TRAIL (VA/TRAIL), it triggered significant apoptosis, causing little or no toxicity in normal mouse splenocytes. VA/TRAIL-induced cell death is involved in the loss of mitochondrial transmembrane potential and the consequent activation of caspases. Because nuclear factor (NF)-κB inhibition has been known as a critical target in TRAIL-mediated apoptosis, we also investigated the role of NF-κB in VA/TRAIL treatment. We found that VA upregulated the DNA binding activity of NF-κB, but that the antioxidants glutathione and N-acetyl-l-cysteine, as well as NF-κB inhibitor MG132, and mutant-IκB (m-IκB) transfection, significantly downregulated VA/TRAIL-induced cell death by inhibiting caspase-3 and NF-κB activities. Transfection of mutant-eIF2α also resulted in a decrease in VA/TRAIL-induced cell death by inhibiting of caspase-3, but not NF-κB activity.

Verrucarin A (VA), a protein synthesis inhibitor, derived from the pathogen fungus Myrothecium verrucaria, inhibits growth of leukemia cell lines and activates caspases and apoptosis and inflammatory signaling in macrophages. We have investigated VA-induced growth inhibition in breast cancer cells MDA-MB-231 and T47D and, particularly, the mechanism of VA-induced apoptosis. VA treatment brought about apoptotic cell death in a dose- and time-dependent manner which was associated with chromatin condensation, cell shrinkage, nuclear fragmentation and intracellular ROS production. Mitochondrial membrane depolarization, activation of caspase-3, down-regulation of Bcl-2 expression and up-regulation of Bax and p53 expression were observed. VA thus affects the viability of both the breast cancer cells by triggering ROS-mediated intrinsic mechanism of apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is one of the most promising candidates for new cancer therapeutics. However, resistance to TRAIL in some cancers remains a current problem in recent. The protein-folding compartment of the endoplasmic reticulum (ER) is particularly sensitive to disturbances, which, if severe, may trigger apoptosis. Therefore, we examined whether verrucarin A (VA) sensitize TRAIL-induced apoptosis in cancer cells by induction of ER stress. We first found that VA induces a major molecule of ER stress, CCAAT/enhancer binding protein homologous protein (CHOP)-dependent DR5 induction and subsequently increases TRAIL-induced cleavage of caspases and PARP in TRAIL-resistant Hep3B cells. Importantly, the transient knockdown using siRNA for CHOP abrogated VA-induced DR5 expression and attenuated TRAIL-induced apoptosis. Treatment with VA also increased the levels of phosphorylation of eukaryotic translation initiation factor-2α (eIF2α), which is a common cellular response of ER stress.

The present study was carried out to elucidate the mechanisms underlying Verrucarin A (VA)-induced cytotoxicity in human breast cancer cell line MDA-MB-231. VA inhibited the growth of MDA-MB-231 cells by induction of reactive oxygen species (ROS)-dependent mitochondrial apoptosis. Elevation of ROS production, associated with changes in Bax/Bcl-2 ratio, led to loss of mitochondrial membrane potential (Δψm) and cytochrome c release in VA-treated cells. Release of cytochrome c from mitochondria to cytosol triggered activation of caspase-3, PARP cleavage, DNA fragmentation, and finally apoptotic cell death. Furthermore, VA-induced apoptosis was accompanied by the activation of p38MAPK and inhibition of phosphorylation of EGFR as well as of Akt and ERK1/2. However, pre-treatment with n-acetyl cysteine, an ROS scavenger, and SB202190, a p38MAPK inhibitor, significantly inhibited VA-induced ROS generation, EGFR inhibition, p38MAPK activation and apoptosis.