Z-ATAD-FMK

The search for novel and more efficient chemo-agents against malignant osteoblastoma is important. In this study, we examined the potential anti-osteoblastoma function of bufotalin, and studied the underlying mechanisms. Our results showed that bufotalin induced osteoblastoma cell death and apoptosis in dose- and time-dependent manners. Further, bufotalin induced endoplasmic reticulum (ER) stress activation in osteoblastoma cells, the latter was detected by the induction of C/EBP homologous protein (CHOP), phosphorylation of inositol-requiring enzyme 1 (IRE1) and PKR-like endoplasmic reticulum kinase (PERK), as well as caspase-12 activation. Conversely, the ER stress inhibitor salubrinal, the caspase-12 inhibitor z-ATAD-fmk as well as CHOP depletion by shRNA significantly inhibited bufotalin-induced osteoblastoma cell death and apoptosis. Finally, by using a mice xenograft model, we demonstrated that bufotalin inhibited U2OS osteoblastoma cell growth in vivo. In summary, our results suggest that ER stress contributes to bufotalin-induced apoptosis in osteoblastoma cells. Bufotalin might be investigated as a novel anti-osteoblastoma agent.

Toxoplasma gondii is a major cause of congenital brain disease; however, the underlying mechanism of neuropathogenesis in brain toxoplasmosis remains elusive. To explore the role of T. gondii in the development of neural stem cells (NSCs), NSCs were isolated from GD14 embryos of ICR mice and were co-cultured with tachyzoites of T. gondii RH strain. We found that apoptosis levels of the NSCs co-cultured with 1×106 RH tachyzoites for 24 and 48 h significantly increased in a dose-dependent manner, as compared with the control. Western blotting analysis displayed that the protein level of C/EBP homologous protein (CHOP) was up-regulated, and caspase-12 and c-Jun N-terminal kinase (JNK) were activated in the NSCs co-cultured with the parasites. Pretreatment with endoplasmic reticulum stress (ERS) inhibitor (TUDCA) and caspase-12 inhibitor (Z-ATAD-FMK) inhibited the expression or activation of the key molecules involved in the ERS-mediated apoptotic pathway, and subsequently decreased the apoptosis levels of the NSCs induced by the T. gondii. The findings here highlight that T. gondii induced apoptosis of the NSCs through the ERS signal pathway via activation of CHOP, caspase-12 and JNK, which may constitute a potential molecular mechanism responsible for the cognitive disturbance in neurological disorders of T. gondii.

7-Ketocholesterol (7kCh) is a major oxysterol found associated with vascular diseases. Human microvascular endothelial cells (HMVECs) were cultured with different concentrations of 7kCh with and without inhibitors. Cell viabilities and caspase activities were assessed. 7kCh caused loss of cell viability in a dose-dependent manner. Caspases-8, -12, and -3/7 but not caspase-9 were activated by 7kCh treatment. The 7kCh-induced caspase-8 activity was blocked partially by pre-treatment with z-VAD-fmk and z-IETD-fmk, a caspase-8 inhibitor. However, pre-treatment with z-ATAD-fmk, a caspase-12 inhibitor, followed by 7kCh exposure lead to significantly increased caspase-8 activity. This suggests that caspase-8 and caspase-12 pathways have unique inhibition patterns and that caspase-12 is likely not upstream and feeding into caspase-8 but the pathways may function in parallel to each other. Caspase-3/7 activation was inhibited partially by low density lipoprotein (LDL), high density lipoprotein (HDL), z-VAD-fmk (pan-caspase inhibitor), and low doses (0.01 and 0.001 microM) of the cholesterol lowering drug, simvastatin. However, only LDL partially protected against 7kCh-induced loss of cell viability suggesting that caspase-independent pathways also contributed to the cell loss and that protection from oxysterol damage may require inhibition of multiple pathways. Moreover, our data suggest that oxysterols such as 7kCh can damage HMVECs cells in part via caspase-dependent apoptosis and may play a role in vascular and retinal diseases.