Polyphyllin VII Promotes Apoptosis and Autophagic Cell Death via ROS-Inhibited AKT Activity, and Sensitizes Glioma Cells to Temozolomide

Abstract

The high recurrence frequency of gliomas but deficiency of effective treatment and prevalent chemoresistance have elicited interests in exploring and developing new agents. Paris polyphyllins are monomers extracted from rhizome of Paris polyphylla var. yunnanensis. Here, we first reported that polyphyllin VII (PP7) exhibited cytotoxic effect on glioma cells. PP7 significantly suppressed the viability and induced cell death of U87-MG and U251 cells after 24 h, with the IC50 values 4.24 ± 0.87 μM and 2.17 ± 0.14 μM, respectively. Both apoptotic and autophagic processes were involved in the cytotoxic effect of PP7, as PP7 activated the Bcl2/Bax pathway and the inhibition of autophagy partly rescued the toxicity of PP7 in glioma cells. In addition, an inhibition of AKT/mTORC1 activity was found after PP7 administration, and it seemed that the overproduction of reactive oxygen species (ROS) was responsible for this effect. Namely, the removal of ROS by NAC treatment mitigated PP7-induced cell death, autophagy, and its effect on the AKT/mTORC1 signaling. Additionally, a combination assay of PP7 with temozolomide (TMZ), the most used chemotherapy for glioma patients, was performed resulting in synergism, while PP7 reduced TMZ resistance through inhibition of MGMT expression. Thus, our study reports PP7 as a potential agent for glioma treatment and reveals its underlying mechanisms of action.

Figure 1

PP7 cytotoxicity in U87-MG and U251 cells. (a, b) The cell viability of U87-MG and U251 cells treated with different concentrations of PP7 was assessed after 12, 24, and 36 h. (c–e) Representative images and quantifications display increased cell death of U87-MG and U251 cells under PP7 treatment for 24 h, labeled with Hoechst33342/PI. Arrows indicate dead cells. (f, g) Western blots and their quantification show increased Bax and cleaved caspase-3 protein levels but decreased Bcl-2 levels in a dose-dependent manner in U87-MG cells and (h, i) in U251 cells. (j, k) Quantifications of the ratio of cleaved caspase-3 to total caspase-3 in U87-MG and U251 cells after PP7 treatment. (l, m) Western blots and their quantification show increased Bax protein levels in mitochondria as well as increased cytochrome C protein levels in cytoplasm after PP7 treatment in U87-MG cells and (n, o) in U251 cells. Prohibitin was selected as loading control for mitochondrial fraction, while β-actin was an internal control for the cytoplasmic fraction. Solvent controls are presented as 0 μM groups, while N stands for the repetition of experiments. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 2

PP7 promotes ROS production in U87-MG and U251 cells. (a–c) Representative images and quantification analysis of PP7 effect on ROS production in U87-MG and U251 cells, assessed by dihydroethidium labeling (a, left) and clearance of ROS after NAC treatment (a, right). (d, e) Quantification of CCK-8 assay shows that NAC administration increases cell viability of PP7-treated U251 and U87-MG cells. Ctr represents cells treated with solvent, while N stands for the repetition of experiments. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 3

PP7 induces autophagy in U87-MG and U251 cells. (a–c) Western blots and their quantification show PP7 concentration-dependent decreased SQSTM1 (p62) protein levels and increased LC3II levels accompanied with the increase in LC3 II/LC3 I ratio in U251 cells as well as (d–f) in U87-MG cells. Solvent-treated cells are presented as the 0 μM control groups. (g–i) Western blots and their quantification show time-dependent decreased SQSTM1 (p62) protein levels and increased LC3II levels accompanied with the increase in LC3 II/LC3 I ratio in U251 cells treated with 4 μM PP7 as well as (j–l) in U87-MG cells treated with 3 μM PP7. (m–p) Representative images and quantification analysis display that PP7 promotes the formation of GFP-LC3 puncta in U251 and U87-MG cells, and this process could be inhibited by NAC treatment. Ctr stands for the cells treated with solvent. Arrows indicate the LC3 puncta. N stands for the repetition of experiments. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 4

Autophagy is involved in the antiglioma effect of PP7. (a–c) Western blots and corresponding quantification analysis show the effect of 3-MA in respect to increased SQSTM1 (p62), decreased LC3II protein levels, and the decrease in LC3 II/LC3 I ratio in U87-MG cells and (d–f) in U251 cells. U87-MG and U251 cells were subsequently treated with 3 μM PP7 and 3 μM PP7, respectively, and with 5 mM 3-MA. 3-MA was added 3 h after adding PP7, while the overall treatment lasted 8 h. (g–i) Representative images and quantification analysis display decrease in LC3 puncta after PP7 and 3-MA combined treatment in U251 and U87-MG cells. Ctr represents cells treated only with solvent. (j, k) Cytotoxic effect induced by PP7 was partly rescued by 3-MA treatment in U87-MG and U251 cells. U87-MG and U251 cells were treated with 6 μM and 4 μM PP7, respectively, alone or in combination with 3-MA (5 mM) for 24 h. (l, m) Cytotoxic effect induced by PP7 was partly rescued by Baf-A1 treatment in U87-MG and U251 cells. U251 and U87-MG cells were treated with 4 μM and 6 μM PP7, respectively, alone or in combination with bafilomycin A1 (10 nM) for 24 h. N stands for the repetition of experiments. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 5

PP7 inhibits the AKT/mTORC1 pathway in U251 and U87-MG cells. (a, b) Western blots and corresponding quantification analysis show decreased phosphorylation levels of AKT in PP7-treated U251cells. Solvent-treated cells are presented as 0 μM control. (c, d) Western blots and corresponding quantification analysis show the effects of growth factors' application on phosphorylation levels of AKT after PP7 treatment in U251cells. (e, f) Western blots and corresponding quantification analysis show decreased phosphorylation levels of mTORC1 effectors, 4E-BP1, p70S6K, and S6, in U251 cells treated with PP7. (g, h) Western blots and corresponding quantification analysis show that PP7 inhibits the AKT/mTORC1 pathway in U87-MG. Solvent-treated cells are represented as 0 μM control. (i, j) Western blots and corresponding quantification show that the NAC administration recovers the phosphorylation levels of AKT and mTORC1 effectors induced by PP7 treatment in U251cells. N stands for the repetition of experiments. ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 6

Synergistic cytotoxic effect of PP7 and TMZ combination in U251 and U87-MG cells. (a, b) Cell viability of U251 cells treated with ineffective concentrations of PP7 or TMZ separately for 24 h (N = 3). (c, d) Cell viability of U251 cells treated with combinations of PP7 and TMZ for 24 h (N = 4). (e, f) Cell viability of U87-MG cells treated with ineffective concentrations of PP7 or TMZ separately for 24 h (N = 3). (g, h) Cell viability of U87-MG cells treated with combinations of PP7 and TMZ for 24 h (N = 4). (i, j) Representative images and quantification analysis show that low concentration of PP7, combined with TMZ, induced cell death in U251 cells. Solvent controls are represented as 0 μM. ^∗p < 0.05, ^∗∗∗p < 0.001. (k, l) Representative images and quantification analysis show that low concentration of PP7, combined with TMZ, induced cell death in U87-MG cells. Solvent controls are represented as 0 μM. N stands for the repetition of experiments. ^∗p < 0.05, ^∗∗∗p < 0.001.

Figure 7

PP7 reduces TMZ resistance in U251 and U87-MG cells by suppressing the expression of MGMT. (a) Quantified mRNA expression of MGMT after TMZ treatment in U251 cells (N = 3). (b) PP7 suppresses the expression of MGMT induced by TMZ treatment in U251 cells (N = 3). (c) Quantified mRNA expression of MGMT after TMZ treatment in U87-MG cells (N = 3). (d) PP7 suppresses the expression of MGMT induced by TMZ treatment in U87-MG cells (N = 3). (e, f) Western blots and corresponding quantification analysis show that TMZ activates the phosphorylation of AKT, which could be inhibited by PP7 in U251 cells (N = 3). (g, h) Western blots and corresponding quantification show that TMZ activates the phosphorylation of AKT, which could be inhibited by PP7 in U87-MG cells (N = 3). Solvent controls are represented as 0 μM. N stands for the repetition of experiments. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 8

Schematic illustration of signals involved in cytotoxicity induced by PP7 (left) and by combination of PP7 with TMZ (right).