treated tumors displayed a reduction in MVD that was not different than rapamycin alone suggesting Decitabine that, at least in this tumor model, the antiangiogenic effect observed was due to mostly to rapamycin administration. To further understand the dynamics of rapamycin and enzastaurin treatment in vivo, CAL27 xenografts were established in nude mice, harvested at 3, 7, 10, and 14 days and assayed for phospho Akt . Interestingly, rapamycin treatment was associated with increased phospho Akt expression at all time points, whereas enzastaurin treatment led to a modest decrease at day 14. Furthermore, because MVD was not reduced at day 14 in enzastaurintreated mice we determined whether angiogenesis was affected at any point during treatment.
In fact, at day 3, MVD is significantly reduced in all treatment groups as evidenced by CD31 staining . DISCUSSION Deregulation of multiple signaling pathways and processes occurs in most epithelial cancers. Therefore, we hypothesized that combining agents with different and potentially complementary mechanisms of action would result in superior inhibition of tumor growth than either agent Clofarabine clinical trial alone. Indeed, our in vitro and in vivo results confirm that enzastaurin and rapamycin are more potent in combination with respect to cell viability, induction of apoptosis, and suppression of tumor growth. Moreover, the agents demonstrated inhibition of their respective putative targets, suggesting that the underlying efficacy of the combination is linked to disruption of different yet interacting pathways.
These pathways, in turn, affect multiple biologic processes and in the case of CAL27 cells we observed a consistent increase in apoptosis. It is possible that target inhibition in SCC61 and SQ20B cells is not directly linked to the apoptotic machinery or other prosurvival signals overcome Clofarabine structure apoptosis induction. A feedback activation of Akt on exposure to rapamycin or one of its analogues has been linked with treatment resistance in many cancer systems, including SCCHN.14 In fact, this formed the rationale for combining rapamycin with enzastaurin. We observed evidence of reciprocal Akt activation in the rapamycin treated CAL27 xenografts on the basis of an increase in phosphorylation of a downstream pharmacodynamic marker, GSK3b, and Akt itself. Despite the higher levels of Akt activation, in these tumors, rapamycin was effective as a single agent in the CAL27 xenograft model.
However, enzastaurin did not abrogate this increase in phospho Akt although phospho GSK3b was significantly lower in enzastaurin treated tumors. Taken together, Clofarabine solubility these data suggest that mTOR inhibition exerts antineoplastic effects that overcome the reciprocal activation of Akt and that the mechanism underlying combinatorial synergy of rapamycin and enzastaurin is unrelated to Akt inhibition. We note that rapamycin health insurance treated tumors demonstrated significantly decreased MVD and increased apoptosis compared with controls and addition of enzastaurin produces a reduction in tumor size compared with either single agent alone. The fact that we observed a significant inhibition of angiogenesis as measured by MVD in enzastaurintreated tumors early but not at the conclusion of the experiment was surprising because enzastaurin demonstrates antiangiogenic.