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

Abstract

Purpose: Aberrant activation of protein kinase Ch (PKCh) by pancreatic cancer cells facilitates angiogenesis and tumor cell survival. Targeting PKCh with enzastaurin, a well-tolerated drug in clinical trials, would be expected to radiosensitize pancreatic tumors through direct antitumor and antivascular effects. Experimental Design: We tested the hypothesis that enzastaurin radiosensitizes pancreatic cancer cells in culture and in vivo through inhibition of PKCh. 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 3h, a mediator of cell death in culture and in vivo. Results: At concentrations attained in patients, enzastaurin reduced levels of active PKCh 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 3h at Ser 9 in pancreatic cancer cells in culture and in tumor xenografts, suggesting a possible mechanism for the observed radiosensitization. Conclusions: Enzastaurin inhibits PKCh 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.

Antitumor activity of enzastaurin as radiation sensitizer in head and neck squamous cell carcinoma

“…These indicate that the radiosensitizing effect of enzastaurin on HNSCC cells may be carried out through inhibition of the expression of radioresistance-associated oncoproteins, thereby sensitizing the cells to radiation and resulting in cell death. Consistent with our observation, Spalding et al 19 found that enzastaurin in combination with 2 Gy of radiation can significantly inhibit both GSK3b and PKC b phosphorylation very shortly after adding enzastaurin in pancreatic cancer cell lines BxPC3 and Panc1 and xenograft.…”

“…It has been found that enzastaurin can dramatically suppress the growth of new vasculature in the rat corneal micropocket 20 and results in decreased microvessel in the Panc1 pancreatic cell line. 19 To investigate the antivascular effects of enzastaurin, we stained mice tumor tissue for CD31 to determine its effect on blood vessel density of each treatment group. We found that enzastaurin alone and enzastaurin in combination with radiation both resulted in decreased blood vessel density compared with no-treatment control, and the combination treatment was most effective at inhibiting blood vessel formation and also induced tumor tissue necrosis; whereas radiation alone was not obviously different from control except for more fibrosis.…”

Protein Kinase C (PKC) Isozymes and Cancer

“…Treatment with the PKC inhibitor enzastaurin reduces cancer growth, phosphorylation of GSK3 , and microvessel density in pancreatic cancer-bearing mice when combined with radiation therapy, but enzastaurin alone failed to affect cell survival, proliferation, or xenograft growth [413,414]. On the other hand, in a study using the pancreatic endocrine cancer cell line DON1, enzastaurin (5 and 10 M) increased caspasemediated apoptosis, reduced phosphorylation of GSK3 and Akt, and blocked cell proliferation through the inhibition of insulin-like growth factor-1 (IGF-1) [415].…”

Molecular pathways involved in the synergistic interaction of the PKCβ inhibitor enzastaurin with the antifolate pemetrexed in non-small cell lung cancer cells

“…Enzastaurin was originally evaluated in human tumour xenograft-bearing mice for its antiangiogenic activity upon PKCb inhibition, as it showed reduction of plasma VEGF levels together with a significant decrease in intratumoural vessel density (Keyes et al, 2004). However, several studies have shown that enzastaurin exhibits direct growth inhibiting effects on a wide array of cultured human tumour cells (Graff et al, 2005;Oberschmidt et al, 2005;Querfeld et al, 2006;Rizvi et al, 2006;Podar et al, 2007;Spalding et al, 2007;Lee et al, 2008). Recent studies suggest that the antitumour effects of enzastaurin are mediated through interference with the phosphatidylinositol 3-kinase (PI3K)/Akt pathway (Graff et al, 2005;Querfeld et al, 2006;Rizvi et al, 2006;Lee et al, 2008), an important pathway regulating the apoptotic response.…”