Targeting protein kinase C by Enzastaurin restrains proliferation and secretion in human pancreatic endocrine tumors

Abstract

Dysregulation of the protein kinase C (PKC) signaling pathway has been implicated in tumor progression. In this study, we investigate the effects of a PKC inhibitor, Enzastaurin, in human pancreatic neuroendocrine neoplasms (PNN) primary cultures and in the human pancreatic endocrine cancer cell line, BON1. To this aim six human PNN dispersed in primary cultures and BON1 cells were treated without or with 1-10 mM Enzastaurin and/or 100 nM IGF1 in the presence or absence of serum. Cell viability and apoptosis were evaluated after 48-72 h; Chromogranin A (CgA) and/or insulin secretion was assessed after 6 h of incubation. PKC expression was investigated by immunofluorescence and western blot. We found that Enzastaurin significantly reduced human PNN primary culture cell viability, as well as CgA and insulin secretion. Moreover, in the BON1 cell line Enzastaurin inhibited cell proliferation at 5 and 10 mM by inducing caspasemediated apoptosis, and reduced phosphorylation of glycogen synthetase kinase 3b (GSK3b) and of Akt, both downstream targets of PKC pathway and pharmacodynamic markers for Enzastaurin. In addition, Enzastaurin blocked the stimulatory effect of IGF1 on cell proliferation, and reduced CgA expression and secretion in BON1 cells. Two different PKC isoforms are expressed at different levels and have partially different subcellular localization in BON1 cells. In conclusion, Enzastaurin reduces cell proliferation by inducing apoptosis, with a mechanism likely involving GSK3b signaling, and inhibits secretory activity in PNN in vitro models, suggesting that Enzastaurin might represent a possible medical treatment of human PNN.

Identification of Candidate Serum Proteins for Classifying Well-Differentiated Small Intestinal Neuroendocrine Tumors

“…IGF1 is a protein similar to insulin in function and structure and is a member of a protein family involved in mediating growth and development [20]. The activation of the IGF1/IGF1 receptor system (IGF1/IGF1R) is a critical event in transformation and tumorigenesis in a wide variety of human tumors [21]-[23].…”

mTOR inhibitors response and mTOR pathway in pancreatic neuroendocrine tumors

“…Tissue samples were stored in RNA-later solution (Sigma) for expression studies; upon arrival in the lab, they were immediately frozen in liquid nitrogen under ribonuclease (RNase)-free conditions and stored at −80°C until protein isolation was performed. A portion of the fresh tissue was collected in culture medium for primary culture studies; upon arrival in the lab, the tissue was immediately processed for primary culture experiments as described previously, with minor modifications (Molè et al 2011). Tumor cells were resuspended in F-12 with 10% fetal bovine serum (FBS) and antibiotics (Euroclone Ltd, Wettherby, UK), seeded at 2 × 10 4 cells/well in 96-well black plates and incubated at 37°C in a humidified atmosphere of 5% CO 2 -95% air, as described previously (Zatelli et al 2010a).…”

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].…”