Inhibition of Protein Kinase Cβ by Enzastaurin Enhances Radiation Cytotoxicity in Pancreatic Cancer

Abstract

Aberrant activation of protein kinase Cbeta (PKCbeta) by pancreatic cancer cells facilitates angiogenesis and tumor cell survival. Targeting PKCbeta with enzastaurin, a well-tolerated drug in clinical trials, would be expected to radiosensitize pancreatic tumors through direct antitumor and antivascular effects. We tested the hypothesis that enzastaurin radiosensitizes pancreatic cancer cells in culture and in vivo through inhibition of PKCbeta. We analyzed pancreatic cancer xenografts for growth delay and microvessel density after treatment with enzastaurin, radiation, or both. We determined the effect of radiation and enzastaurin on glycogen synthase kinase 3beta, a mediator of cell death in culture and in vivo. At concentrations attained in patients, enzastaurin reduced levels of active PKCbeta measured by phosphorylation at Thr(500) in culture and in xenografts. Enzastaurin alone did not affect pancreatic cancer cell survival, proliferation, or xenograft growth. However, enzastaurin radiosensitized pancreatic cancer cells in culture by colony formation assay. Enzastaurin alone decreased microvessel density of pancreatic cancer xenografts without appreciable effects on tumor size. When combined with radiation, enzastaurin increased radiation-induced tumor growth delay with a corresponding decrease in microvessel density. Enzastaurin inhibited radiation-induced phosphorylation of glycogen synthase kinase 3beta at Ser(9) in pancreatic cancer cells in culture and in tumor xenografts, suggesting a possible mechanism for the observed radiosensitization. Enzastaurin inhibits PKCbeta in pancreatic cancer cells in culture, enhancing radiation cytotoxicity. Additional antivascular effects of enzastaurin were observed in vivo, resulting in greater radiosensitization. These results provide the rationale for a clinical trial in locally advanced pancreatic cancer combining enzastaurin with radiation.