Even though these caspase inhibitors are extensively utilised for cell-based assay at concentrations of up to 20100 ?M, the in vitro caspase action assay has shown that the caspase inhibitors possess cross-reactivity toward non-targeted caspases at a concentration of one ?M, and just about every of them induce total inhibition of caspase-3, -7, and -8 actions at a greater concentration of 10 ?M. In our hands, having said that, the minimal concentration of z-VAD-fmk, z-LEHD-fmk, or z-DEVD-fmk to thoroughly avert mollugin-induced apoptosis of Jurkat T cells was ?30 ?M, since mollugin-induced apoptotic sub-G1 peakwas not abrogated by pretreatmentwith z-VADfmk, z-LEHD-fmk, or z-LEVD-fmk at concentrations of as much as 20 ?M .
However, the minimum concentration of your caspase-12 inhibitor z-ATAD-fmk to prevent the mollugin-induced apoptosis appeared to become ?4 ?M, considering that selleck chemical extra resources mollugin-induced apoptotic sub-G1 peak was fully abrogated at a concentration of four ?M, but not at concentrations of as much as 2 ?M . Since the in vitro caspase-12 action assay using the cell lysate of J/Neo cells exposed to mollugin revealed that though z-ATAD-fmk could specifically inhibit the caspase-12 action by ?50%, it was very likely the inhibitory effect of z-ATAD-fmk about the mollugin-induced apoptotic signaling pathway was brought about by its exact inhibition towards the induced caspase-12 exercise. To examine if there is a big difference during the apoptogenic result ofmollugin on tumor cells and ordinary cells, we’ve compared the cytotoxicity of mollugin towards leukemia Jurkat T cells with that towards typical human T cells.
The IC50 worth for resting human T cells, activated T cells, Doxorubicin and Jurkat T cells appearedto be N30 ?M,?thirty ?M, and 23 ?M, respectively. This indicated that normal human T cells, specifically, while in the resting state have been extra refractory on the cytotoxicity of mollugin as in contrast with Jurkat T cells, which may permit the greater application of mollugin to chemotherapeutic treatments. Given that the cytotoxicity of mollugin in T cells was attributable to apoptotic cell death provoked by ER stress-mediated activation of JNK and caspase-12, and subsequent mitochondria-dependent activation of caspase cascade, it was very likely the most effective resistance of unstimulated peripheral T cells, as in contrast with other cells tested against mollugin, may be attributable to a poorly produced endoplasmic reticulum and mitochondria, plus a lowlevel of death signalingmediators in unstimulated peripheral T cells.
Then again, the exact molecular mechanism underlying the ideal resistance of unstimulated T cells to mollugin-induced apoptosis even now stays to become elucidated.