Taurine 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 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 phospholipases 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.
The dual therapy treated tumors displayed a reduction in MVD that berberine was not different than rapamycin alone suggesting 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 xenograftswere 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 MK-8669 growth than either agent 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 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.