Sunitinib induces apoptosis in pheochromocytoma tumor cells by inhibiting VEGFR2/Akt/mTOR/S6K1 pathways through modulation of Bcl-2 and BAD

Abstract

Sunitinib is an oral multitargeted receptor tyrosine kinase inhibitor with antiangiogenic and antitumor activity that mainly targets vascular endothelial growth factor receptors (VEGFRs). Very recently, sunitinib has been shown to be an active agent for the treatment of malignant pheochromocytomas. However, it is unclear whether sunitinib acts only through an antiangiogenic mechanism or whether it may also directly target tumor cells. Sunitinib markedly induced apoptosis of PC12 cells in a dose-dependent and time-dependent manner. Furthermore, in support of these findings, we found that sunitinib induced a reduction in the expression of the antiapoptotic molecule Bcl-2 as well as dephosphorylation of the proapoptotic molecule BAD, which results in the activation of BAD in these cells. Consistent with these apoptotic effects, our results showed that sunitinib inhibited phosphorylation of Akt and mTOR and was followed by a reduction of S6K1, which is a well-known target of mTOR. Knockdown of VEGFR-2 attenuated the sunitinib-induced effects, including apoptosis and inhibition of signaling pathways such as the phosphorylation of Akt as well as mTOR, and Bcl-2, which confirmed that these effects could be mediated by VEGFR-2. In addition, silencing of S6K1 induced apoptosis accompanied by a decrease in the phosphorylation of BAD and Bcl-2, similar to that observed with sunitinib treatment. Thus, these results together suggest that sunitinib initially exerts its apoptotic effect through the inhibition of VEGFR-2, which, when followed by reduction of its downstream effectors, including Akt/mTOR/S6K1, may lead to inhibition of the antiapoptotic molecule Bcl-2 and activation of the proapoptotic molecule BAD in PC12 cells. However, PC12 cells do not precisely reflect the pathogenesis of malignant cells. Therefore, we confirmed the key findings by replicating these experiments in human neuroblastoma SK-N-SH cells.

Interferon alpha and rapamycin inhibit the growth of pheochromocytoma PC12 line in vitro

“…These facts suggest that VEGF did not affect PC12 cell growth directly, but indirectly contributed to the growth of xenografted PC12 tumour in vivo by stimulation of angiogenesis. These observations can also explain the fact that in our experiments endostatin, which acts mainly by blocking VEGF/VEGFR signalling [38], does not have any effect on the growth of PC12 cell line. Moreover, other authors did not show a direct binding of endostatin to the PC12 cell surface [28,39], suggesting the absence of receptors for endostatin in this cell line.…”

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

“…Very recently, sunitinib was proven to be an active agent in the treatment of malignant pheochromocytomas (3 -5). Previously, we demonstrated that sunitinib directly inhibited mTORC1 signaling, which in turn led to apoptosis in PC12 cells (6). This indicated that sunitinib may exert its antitumor effects, at least in part, directly via tumor cells rather than by antiangiogenesis.…”

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.…”

“…With only three studies on the topic, however, it is impossible to generalize the results to any useful effect. Furthermore, all of the studies were conducted in vitro on cell lines that, in the case of the rat PC12 cells, are known to imprecisely represent malignant pheochromocytoma cells (Saito et al 2012). Future research is required to elucidate the specifics of autophagy in the different types of adrenal cancer in vitro and in vivo and take full advantage of its therapeutic potential as a target for induction or inhibition, as indicated.…”