Piperlongumine promotes autophagy via inhibition of Akt/mTOR signalling and mediates cancer cell death

Abstract

Background: The Akt/mammalian target of rapamycin (mTOR) signalling pathway serves as a critical regulator of cellular growth, proliferation and survival. Akt aberrant activation has been implicated in carcinogenesis and anticancer therapy resistance. Piperlongumine (PL), a natural alkaloid present in the fruit of the Long pepper, is known to exhibit notable anticancer effects. Here we investigate the impact of PL on Akt/mTOR signalling.

Methods: We examined Akt/mTOR signalling in cancer cells of various origins including prostate, kidney and breast after PL treatment. Furthermore, cell viability after concomitant treatment with PL and the autophagy inhibitor, Chloroquine (CQ) was assessed. We then examined the efficacy of in vivo combination treatment using a mouse xenograft tumour model.

Results: We demonstrate for the first time that PL effectively inhibits phosphorylation of Akt target proteins in all tested cells. Furthermore, the downregulation of Akt downstream signalling resulted in decrease of mTORC1 activity and autophagy stimulation. Using the autophagy inhibitor, CQ, the level of PL-induced cellular death was significantly increased. Moreover, concomitant treatment with PL and CQ demonstrated notable antitumour effect in a xenograft mouse model.

Conclusions: Our data provide novel therapeutic opportunities to mediate cancer cellular death using PL. As such, PL may afford a novel paradigm for both prevention and treatment of malignancy.

Figure 1

Piperlongumine affects Akt downstream signalling in cancer cells of various origins. (A) Piperlongumine decreases phosphorylation levels of Akt effectors, GSK-3β and TSC2. Additionally, PL treatment results in significant downregulation of mTORC1 complex activity but did not affect the main components of mTORC1 complex, mTOR and Raptor. Cells were treated with PL at indicated concentrations for 2, 4, 8 h or overnight (ON, 20–24 h). (B) Western blot analysis of PTEN expression in 786-O, PC-3 and MCF-7 cells. (C) The inhibitory effect of PL on functioning of constitutively active Akt(T308D; S473D) mutant in MCF-7 cells. MCF-7-HA-Akt^mut cells were treated with PL at indicated concentrations for 24 h. Total cellular lysates were subjected to western blotting with the specific antibodies.

Figure 2

Piperlongumine demonstrates antiproliferative effect in 786-O (A) and MCF-7 (B) cells. Cells were treated with indicated concentrations of PL for 48 h. Cellular proliferation was assessed using the CellTiter Blue assay.

Figure 3

Treatment of 786-O, PC-3 and MCF-7 cells with PL results in increased intracellular ROS production, whereas addition of NAC completely blocks this process. Cells were treated with PL and/or NAC for 2 h. Harvested cells were stained with CM-H₂DCFDA and analysed by flow cytometry. The figure shows the representative data of one of three separate experiments (Bars, s.e.m. of triplicates).

Figure 4

NAC reverses negative effects of PL on Akt downstream signalling. 786-O, PC-3 and MCF-7 cells were treated with PL at indicated concentrations alone or in combination with 10 mM of NAC for 24 h. Total cellular lysates were subjected to western blotting with specific antibodies.

Figure 5

Treatment with PL promotes autophagy induction. (A) Piperlongumine treatment results in LC3-II accumulation and decrease of ULK1 Ser757 phosphorylation. Piperlongumine induction of autophagy is ROS dependent, as NAC completely reverses effects of PL. Cells were treated with indicated concentrations of PL alone or in combination with 10 mM of NAC for 24 h. (B) Concomitant treatment of cells with PL (10 μM) and Baf-A1 (10 μM) results in higher accumulation of LC3-II then treatment with PL alone, which indicates PL acts as autophagy inducer rather than inhibitor. Total cellular lysates were subjected to western blotting with specific antibodies. Upper band (if visible) corresponds to LC3-I and lower band corresponds to LC3-II form of LC3 protein.

Figure 6

Immunofluorescent detection of increased autophagosome flux in cells treated with PL. N-Acetyl-L-Cysteine reverses the autophagy-inducing effect of PL. Additionally, cells were treated with mTORC1 inhibitor, temsirolimus, which induced autophagy serving as a positive control. Light chain 3-II is shown in green and DAPI in blue. Bar, 50 μm. The full colour version of this figure is available at British Journal of Cancer online.

Figure 7

Inhibition of autophagy by CQ promotes PL-mediated cancer cell death in vitro. Cells were treated with either 20 μM of CQ alone, with 10 μM of PL alone or concomitantly for 72 h. Cells were then harvested, PI was added to cellular suspensions at 3 μg ml⁻¹ concentration and analysed by Flow cytometry. The representative data from one of three independent experiments are presented.

Figure 8

The concomitant treatment with PL and CQ results in inhibition of tumour growth xenograft mouse tumour model. Subcutaneous PC-3 tumors were established in 6-week-old male C B17/Icr-scid mice. Treatment with PL and/or CQ and assessment of tumor growth were carried out as described in Materials and Methods. Data shown are mean of five mice in each group (s.e.m. displayed with bars).