Apoptosis was also evident by examining the subGG fraction after fluorescence activated cell sorting in HCT cells. These Aspirin analyses show that Calcitriol enzastaurin induces apoptosis in cultured human cancer cell lines in the low micromolar range. Enzastaurin blocks phosphorylation of GSKB and ribosomal protein S.To examine more fully the effects of orally given enzastaurin on the growth of human tumor xenografts, we sought to identify a dose that would yield levels of enzastaurin and its metabolites comparable with what is reached in clinical trials.We chose a dose of mgkg given orally by gavage twice daily.Xenograftbearing mice were treated for consecutive days starting when the mean tumor volume reached f mm.Enzastaurin treatment significantly suppressed the growth of both HCT colon carcinoma. We therefore sought to examine whether pathways known to be influenced by PKC activity might be affected in human tumor cells by enzastaurin treatment.PKC activity has been connected to many intracellular signaling cascades, including the rasERK signaling axis. The time course for enzastaurin plasma exposure was similar on day and on day after dosing. B, with nearly undetectable levels of drug in plasma between and hours after dosing.Consequently, hour time points were not included in further studies.Alternately, these data may simply reflect differences in these xenografted tumors related to tumor content, host tissue content, etc.Enzastaurin suppresses cell signaling within the AKT pathway.Athymic nude mice bearing HCT colon cancer xenografts were treated with mgkg enzastaurin twice daily by gavage after tumors reached a mean tumor volume of mm. Athymic nude mice bearing UMG glioblastomas xenografts were treated with mgkg enzastaurin twice daily by gavage after tumors reached a mean tumor volume of mm. The direct, proapoptotic effects of enzastaurin treatment on human tumor cells were achieved at drug concentrations similar to those achieved in the plasma of clinical trials patients. Based in part on this robust antiangiogenic activity, enzastaurin was advanced into clinical trials as a novel anticancer therapy.We now show that enzastaurin also exhibits a direct effect on human tumor cells, inducing apoptosis in, and suppressing proliferation of, a wide array of cultured human tumor cells.Interestingly, previous work had indicated that enzastaurin treatment suppressed VEGF expression by tumor xenogra fts. VEGF expression is regu lated at both the transcriptional and posttranscriptional levels through activation of the AKT pathway. It is unclear how enzastaurin may interfere with signaling through the PIKAKT pathway. Furthermore, the time course for these signaling changes is different.Collectively, these data suggest that enzastaurin may indirectly suppress signaling through the AKT pathway.The PKC inhibitor PKC has also been shown to suppress AKT pathway signaling, upstream or at the level of AKT, although no direct mechanism was clear in these studies either. Recent evidence has now shown that various PKC family members can regulate AKT activity.Activation of PKCD can also activate AKT in glioblastoma cells, supporting glioblastoma proliferation. At these concentrations, enzastaurin can directly suppress the kinase activity of multiple PKC isoforms.It is therefore conceivable that interference with the AKT signaling pathway may be related to the effect of enzastaurin on multiple PKC family members.

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