PKC activation can trigger signaling through the rasextracellular signalregulated kinase as well as the induction of intestinal cell invasiveness. Recent work has now shown a link between PKC activity and activity of the phosphatidylinositol kinase AKT pathway, a prominent regulatory pathway governing the apoptotic response.Enzastaurin also decreased microvessel density and VEGF expression in human tumor xenografts. The striking antiangiogenic effects of enzastaurin prompted the clinical development of enzastaurin.Oral dosing of enzastaurin to achieve plasma concentrations of drug comparable with those achieved in clinical trials significantly suppresses the growth of human colon and glioblastoma xenografts.Xenograft tumor studies.Mice were monitored daily for palpable tumors.Enzastaurin treatment was initiated when the tumors reached a group mean of mm.The supernatant was then saved for western blotting or ELISA.Briefly, blood from five identically treated mice was pooled into a single CPT tube and centrifuged at, RCF for minutes to separate blood components.The PBMC layer was collected and washed with mL of icecold DPBS, centrifuged minutes at rpm, and resuspended in AL of complete lysis buffer.Briefly, cells were plated per well in a well plate and changed to fresh media with or without enzastaurin on days and. On day, the media were removed and AL propidium iodide were added to each well.The plate was then frozen at jC for hours, thawed, and reread.The proliferative index was scored by subtracting the prefreeze data. Briefly, cells were plated per well in well plates, incubated with or without enzastaurin for to hours. The log volume data are analyzed with a twoway repeatedmeasures ANOVA by time and treatment using SAS PROC MIXED software. PKC activation has now been implicated in tumor cell 5-hydroxytryptophan proliferation and apoptosis as well. We therefore sought to Carvacrol examine whether enzastaurin would also show direct antitumor activity.We evaluated the ability of enzastaurin to suppress tumor cell proliferation in cu lture. Indeed, enzastaurin suppressed the proliferation of UMG glioblastoma cells, PC prostate carcinoma cells, and HCT colon carcinoma cells in the low micromolar range. Evaluation of the NCI cell line panel also showed the antiproliferative activity of enzastaurin in the low micromolar range in a wide variety of cancer cell lines: leukemia, colon cancer, melanoma, renal cancer. The cell lines most sensitive to enzastaurin were K, MOLT, HOP, and PC.Cell lines of the NCI cell line panel that were unaffected by enzastaurin include the prostate cancer cell line DU; the breast cancer cell lines HST, BT, and TD; the melanoma cell line MALMEM; lung cancer cell lines HOP, NCIH, NCIHM, and NCI; the ovarian cancer cell lines OVCAR and SKOV; and the renal cancer cell lines, A, ACHN, RXF, and TK. These data collectively show that enzastaurin suppresses the proliferation of a wide array of cancer cell lines in the low micromolar range, the same concentration range achieved in the plasma of clinical trials patients. We next sought to determine whether enzastaurin might induce apoptosis in tumor cells.As measured by oligonucleosomal fragmentation, enzastaurin induced apoptosis in both HCT colon carcinoma cells and UMG glioblastoma cells in the low micromolar range.