Mice were treated with rapamycin 10 mg/kg once per day, given intraperitoneally; enzastaurin 100 mg/kg thrice per day via oral gavage; enzastaurin thrice and rapamycin once daily at the same doses; or equal volume of diluents. The treatment lasted 14 days. For the time course experiments, 48 mice were xenografted Erlotinib with CAL27 tumors in the right hind limb and randomly divided into 16 groups of 3 animals each. At days 3, 7, 10, and 14, xenografts were harvested from each treatment group of vehicle control, rapamycin, enzastaurin, and rapamycin plus enzastaurin. Differences in tumor growth were compared by use of 1 sided paired t test. Animals from each group were killed for tissue retrieval less than 8 hours after the last treatment.
For each sample, half of the tissue was snap frozen in liquid N2 followed by lysis in radioimmunoprecipitation assay buffer for protein immunoblot analysis. The other half was fixed in 4% buffered formaldehyde overnight, dehydrated, and embedded in paraffin for immunohistochemistry. Lopinavir clinical trial For each sample, the process was finished in less than 20 minutes after sacrifice. Immunohistochemistry and Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling . Tissue samples were processed and embedded in paraffin. The paraffin embedded samples were cut into 4 lm sections. Sections were deparaffinized in xylene, rehydrated through graded alcohols, and transferred to PBS. Immunohistochemistry detection of Ki 67 and CD 31 was performed by use of Ki 67 rabbit monoclonal In Vitro Effects of Rapamycin and Enzastaurin on Putative Targets, Apoptosis, and Cell Viability.
We examined SCCHN cell lines to determine whether rapamycin and enzastaurin inhibited mTOR, PKC, and Akt activation with phosphorylation of p70S6 kinase , myristoylated alanine rich protein kinase c substrate , and glycogen synthase kinase 3 beta as respective substrates . As predicted, rapamycin inhibited phosphop70S6K but had no effect Lopinavir structure on phospho MARCKS or phospho GSK3b. Conversely, enzastaurin reduced phosphorylation of the latter substrates but had minimal effect on phospho p70S6K. The antiproliferative effects of rapamycin and enzastaurin in cancer cell lines have been described, but we sought to determine whether either agent induced apoptosis. FACS analysis with propidium iodide and annexin V staining revealed that the cell lines displayed differential induction of apoptosis .
In CAL27 and SCC61 the number of apoptotic cells was highest when incubated with both agents, but modest to no induction of apoptosis was observed in SQ20B cells even with the combination. We then tested the effect of both agents on SCCHN cell viability and calculated combination Lopinavir solubility indices wealth inequalities with the median effect equation by Chou and Talalay,11 where synergy, additivity, and antagonism are defined by CI <1, 1, and >1, respectively. In all 3 cell lines CI values at 75% inhibition reflected synergy, confirming that the combination of both agents was more effective than either agent alone . Furthermore, CI values at IC50 also indicated synergy . In a broader panel of SCCHN cell lines including ones that are relatively resistant to rapamycin, synergy was also observed with SCC68 a notable exception where maximal concentrations of both agents .