Therefore, the combination of rapamycin and enzastaurin inhibits relevant targets, induces apoptosis, and reduces cell viability in most SCCHN cell lines. In Vivo Effect of Rapamycin and Enzastaurin on Tumor Growth, Putative Targets, Angiogenesis, Temsirolimus Proliferation, and Apoptosis. We observed the most significant effects on apoptosis and cell viability in the CAL27 cell line in vitro and thus tested whether the combination of rapamycin and enzastaurin was more Gefitinib clinical trial effective than either agent alone in vivo. Rapamycin dose was based on reproducibly inhibiting phospho p70S6K in vivo , and the recommended dose and schedule of enzastaurin on the basis of target inhibition and murine pharmacokinetics 12 was administered.
Both enzastaurin and rapamycin inhibited CAL27 tumor growth compared with vehicle treated control, with the latter more effective as a single agent . Interestingly, the combination had the greatest effect on tumor growth with a greater than 70% relative reduction in mean tumor size. Moreover, toward the end of the experiment tumor growth rate in enzastaurin and vehicle treated mice appeared Gefitinib structure to be equivalent, suggesting that enzastaurin was becoming less effective over time but that this could be overcome by rapamycin coadministration. We determined whether the growth inhibition observed in vivo was associated with inhibition of putative enzastaurin targets. After the mice were killed, tumors were harvested and analyzed for expression of phospho MARCKS and phospho GSK3b . Consistent with the in vitro experiments, enzastaurin inhibited both these markers compared to vehicle control.
As expected, rapamycin had no effect on phospho MARCKS expression and Gefitinib solubility consistent with a feedback activation of Akt, phospho GSK3b increased significantly in tumors from rapamycintreated animals. Both rapamycin and enzastaurin have been associated with inhibition of proliferation and apoptosis therefore, we determined whether the same effects could be observed in vivo. Immunohistochemical staining of tumor tissue from treated animals demonstrated a significant reduction in proliferation in all 3 treatment groups as measured by Ki 67 . However, tumors exposed to enzastaurin and rapamycin did not demonstrate any further decrement in Ki 67 staining compared with single agent treatment implicating an alternative process accounting for the greater efficacy of the combination.
TUNEL staining was used to assess the degree of apoptosis present in each condition. A significant increase in apoptosis was observed in all 3 treatment groups and was especially evident with rapamycin alone or rapamycin and enzastaurin . Both rapamycin and enzastaurin have been noted to possess antiangiogenic activity; the former possibly through inhibition of vascular wealth inequalities endothelial growth factor production13 and the latter likely through a direct inhibition of PKCb, which is a downstream mediator of vascular endothelial growth factor induced endothelial cell proliferation.9 Therefore, we stained sections of harvested tumors for the endothelial cell marker, CD31, and calculated microvessel density . As expected rapamycin treated tumors demonstrated a pronounced reduction in MVD . Surprisingly, tumors from enzastaurin treated mice did not demonstrate a change in MVD.