Inhibition of Autophagy Enhances Sunitinib-Induced Cytotoxicity in Rat Pheochromocytoma PC12 cells

Abstract

Abstract. Sunitinib is an oral multitargeted receptor tyrosine kinase inhibitor with antiangiogenic and antitumor activity that mainly targets vascular endothelial growth factor receptors, and recently, it has been shown to be an active agent for the treatment of malignant pheochromocytomas. Previously, we demonstrated that sunitinib directly inhibited mTORC1 signaling in rat pheochromocytoma PC12 cells. Although autophagy is a highly regulated cellular process, its relevance to cancer seems to be complicated. It is of note that inhibition of mTORC1 is a prerequisite for autophagy induction. Indeed, direct mTORC1 inhibition initiates ULK1/2 autophosphorylation and subsequent Atg13 and FIP200 phosphorylation, inducing autophagy. Here, we demonstrated that sunitinib significantly increased the levels of LC3-II, concomitant with a decrease of p62 in PC12 cells. Following sunitinib treatment, immunofluorescent imaging revealed a marked increased punctate LC3-II distribution. Furthermore, Atg13 knockdown significantly reduced its protein level, which in turn abolished sunitinib-induced autophagy. Moreover, inhibition of autophagy by siRNAs targeting Atg13 or by pharmacological inhibition with ammonium chloride, enhanced both sunitinib-induced apoptosis and anti-proliferation. Thus, sunitinib-induced autophagy is dependent on the suppression of mTORC1 signaling and the formation of ULK1/2-Atg13-FIP200 complexes. Inhibition of autophagy may be a promising therapeutic option for improving the anti-tumor effect of sunitinib.

Autophagy in endocrine tumors

“…Unlike RGZ-induced autophagy in adrenocortical carcinoma cells, however, current research suggests that chemically induced autophagy in the rat pheochromocytoma cell line, PC12, promotes cell survival (Ikeda et al 2013, Fabrizi et al 2014. For example, sunitinib, a known anticancer drug, induces both apoptosis and autophagy in PC12 cells, most likely through the direct inhibition of mTOR (Saito et al 2012, Ikeda et al 2013. The inhibition of autophagy, however, increased the apoptotic and antiproliferative effects of sunitinib treatment in PC12 cells (Ikeda et al 2013), implying that in this case autophagy is acting against the anticancer effects of sunitinib and in favor of PC12 cell survival.…”

“…The inhibition of autophagy, however, increased the apoptotic and antiproliferative effects of sunitinib treatment in PC12 cells (Ikeda et al 2013), implying that in this case autophagy is acting against the anticancer effects of sunitinib and in favor of PC12 cell survival. This finding suggests that the targeted inhibition of autophagy may be a therapeutic option for increasing the effectiveness of and surmounting resistance to sunitinib in the treatment of pheochromocytomas (Ikeda et al 2013). Although lithium was also proposed as a treatment option for pheochromocytomas due to its ability to inhibit PC12 cell growth in culture (Kappes et al 2007), the doses used in that paper were well outside the established therapeutic window for lithium use.…”

“…Unlike RGZ-induced autophagy in adrenocortical carcinoma cells, however, current research suggests that chemically induced autophagy in the rat pheochromocytoma cell line, PC12, promotes cell survival (Ikeda et al 2013, Fabrizi et al 2014. For example, sunitinib, a known anticancer drug, induces both apoptosis and autophagy in PC12 cells, most likely through the direct inhibition of mTOR (Saito et al 2012, Ikeda et al 2013.…”

“…For example, sunitinib, a known anticancer drug, induces both apoptosis and autophagy in PC12 cells, most likely through the direct inhibition of mTOR (Saito et al 2012, Ikeda et al 2013. The inhibition of autophagy, however, increased the apoptotic and antiproliferative effects of sunitinib treatment in PC12 cells (Ikeda et al 2013), implying that in this case autophagy is acting against the anticancer effects of sunitinib and in favor of PC12 cell survival. This finding suggests that the targeted inhibition of autophagy may be a therapeutic option for increasing the effectiveness of and surmounting resistance to sunitinib in the treatment of pheochromocytomas (Ikeda et al 2013).…”

“…In the treatment of one adrenocortical carcinoma cell line, RGZ appears to exert its anticancer effects via the induction of autophagy (Cerquetti et al 2011). In the treatment of pheochromocytomas, however, autophagy acts as a protective mechanism for the PC12 cells in the face of cytotoxic treatments (Ikeda et al 2013, Fabrizi et al 2014. Even between two different cell lines of one subtype of adrenal cancer autophagy is differentially involved in treatment mechanism.…”