Puquitinib mesylate (XC-302) induces autophagy via inhibiting the PI3K/AKT/mTOR signaling pathway in nasopharyngeal cancer cells

There are numerous studies that demonstrate the anti-neoplastic activity of phosphatidylinositol 3-kinase (PI3K) inhibitors and the mechanisms of inducing autophagy in cancer cells. The new anticancer drug puquitinib mesylate (XC-302) is a molecular-targeted drug, which suppresses the activity of PI3K directly. However, it remains unclear whether XC-302 can develop an antitumor effect by inducing autophagy in naso-pharyngeal cancer cells. The MTT assay was used to study the anti-proliferative effects of XC-302. Subsequently, autophagy was determined by monodansylcadaverine (MDC) staining, punctate localization of green fluorescent protein (GFP)-light chain 3 (LC3), LC3 protein blotting and electron microscopy. The expression levels of beclin 1, p62, protein kinase B (AKT), phospho (p)-AKT, mechanistic target of rapamycin (mTOR) and p-mTOR in XC-302-induced autophagy were detected. Autophagy inhibition was assayed by 3-methyladenine (3-MA) or small interfering RNA (siRNA) silencing of beclin 1. XC-302 inhibited the viability of CNE-2 in a dose-dependent manner and the IC(50) of 72 h was 5.2 µmol/l. After cells were exposed to XC-302 for 24 h, MDC-labeled autophagolysosomes were evident in CNE-2 cells by fluorescence microscope. Autophagosomes and autolysosomes were identified by transmission electron microscopy. Following transfection with GFP-LC3, XC-302 induced a significant accumulation of GFP-LC3, as monitored by a confocal microscope, which was reduced by 3-MA. XC-302 induced the formation of LC3-II, increased beclin 1 levels and decreased the expression of p62. Additionally, the expression levels of p-AKT and p-mTOR were reduced with the elevation of XC-302. Knockdown of beclin 1 with siRNA or co-treatment with 3-MA enhanced significantly the survival of CNE-2 and promoted the ability of clone formation. XC-302 also induced apoptosis in CNE-2, and when autophagy was inhibited by 3-MA, the apoptosis rate was decreased. The present data provides the evidence that XC-302 can induce autophagy in CNE-2, which promotes the program of cell death and inhibits the PI3K/AKT/mTOR signaling pathway. Furthermore, XC-302 also promoted apoptosis in CNE-2 cells, which could be reduced when autophagy was suppressed, meaning that autophagy may interact with apoptosis to induce cell death.