We further determined whether the differential sensitivity of human cancer cells after a brief exposure of cells to the caspase inhibitor, ZLEHDFMK, and TRAIL would be sustained and evident in a prolonged subsequent culture in the absence of either agent.Thus, we wished to determine whether ultimate SW cell survival, as measured by a colony assay, would be significantly reduced after a brief h exposure to TRAIL in the presence of the caspase inhibitor.The Endurobol results show that colony growth of HCT was reduced in the presence of TRAIL, and there were significantly more colonies present when the HCT cells were incubated in the presence of TRAIL and the caspase inhibitor. As measured by this colony assay, there was a similar number of surviving growing HCT colonies whether TRAIL was coadministered with the caspase inhibitor.However, the combination of the caspase and inhibitors appeared to Imiquimod protect HCT colony growth to a greater extent than either inhibitor alone.In the case of SW cells, the caspase inhibitor significantly preserved colony growth in the presence of TRAIL.Annexin VEGFP staining was performed on the cells in situ.It is possible that caspase activity may in some cells play a more important role, perhaps in feedback amplification of the apoptotic signaling cascade, although we have not specifically investigated this possibility.Caspase, which like caspase is released from the mitochondria, is also activated in both HCT and SW.However, the caspase inhibitor, ZVDAVDFMK, prevents procaspase and PARP cleavage in HCT but not in SW cells.TRAILCASPASE INHIBITOR THERAPY cancer cells will be susceptible to the combination of TRAIL and a caspase inhibitor and which cancer cells would be resistant.In this regard, we have developed a number of in vitro tests using human cancer cells in culture to predict whether the proposed strategy of using TRAIL plus a caspase inhibitor would result in cancer cell death, under conditions in which the normal human hepatocytes are protected from TRAIL.On the basis of these observations, we envision performing similar in vitro testing using viable patient tumor biopsy or surgical specimen material to attempt to predict responsiveness to the combination of TRAIL plus a caspase or other caspase inhibitor.There are efforts to develop caspase inhibitors in the therapy of degenerative neurological diseases. It would not be expected that a brief exposure to a caspase inhibitor would have longterm consequences in terms of, for example, tumorigenicity, but these issues can be further explored in animal studies.It would obviously not be desirable to coadminister a caspase inhibitor at the same time as chemotherapy that relies on caspase for killing cells.However, in these studies, only the general caspase inhibitors ZVADFMK and YVADCMK have been used.The TRAIL ligand is probably more favored for development at present because of the widespread expression of its receptors and the broad sensitivities in many cancer types thus far reported.Little is known, however, about innate host or acquiredresistance to TRAIL in cancer therapy in vivo.In summary, our results provide an indication that suggests a novel strategy using the combination of the caspase inhibitor, ZLEHDFMK, and TRAIL in an effort to maintain the killing effect of many cancer cell types while offering some degree of protection to the human liver.

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