Promotion of Ros-mediated Bax/Cyt-c apoptosis by polyphyllin II leads to suppress growth and aggression of glioma cells

Abstract

Background: Gliomas remain among the most difficult cancers to treat, with a 5-year overall survival no greater than 5%. Many saponins showed a wide spectrum of anti-cancer activities at low concentration. Polyphyllin II is one of the common saponins from Paris polyphylla. However, the effect of Polyphyllin II on glioma cells has not been evaluated. Objective of the present study was to investigate whether Polyphyllin II have inhibition on glioma cells, and the possible mechanisms.

Methods: The viability of U87 and U251 cells was detected by cell counting kit-8, cell counting real time cellular analysis and cell clone formation methods. Transwell was used to estimate the aggression of U87 and U251. The cell apoptosis rate was tested by flow cytometry. The morphological change was determined by transmission electron microscopy. The levels of AKT, phosphorylation of AKT, Bax, Bcl-2, cytochrome c, and cleaved caspase 3 proteins were assessed by Western blot. N-acetyl-L-cysteine was used to check the role of ROS in polyphyllin II inhibition to glioma cells.

Results: Polyphyllin II showed significant suppress to proliferation and aggression of U87 and U251 in a dose- and time- dependent manner. Result of flow cytometry confirmed that Polyphyllin II induced apoptosis to U87 and U251 cells. Transmission electron microscopy observation revealed majority of the glioma cells treated with Polyphyllin II had turgidity of mitochondrion, disarrangement, diminution and vacuolization, those refer to mitochondrial apoptosis. Western blot indicated that Polyphyllin II promoted cyt-c, Bax, caspase 3 and cleaved-caspase 3, and decreased Bcl-2, AKT and p-AKT. Rescue experiments using N-acetyl-L-cysteine, a reactive oxygen species scavenger, reversed the levels of Bax and cyt-c, and the inhibition in Polyphyllin II-treated U87 and U251 cells.

Conclusions: The present findings revealed that polyphyllin II may be a potential drug against glioma.

Keywords: Cancer; Paris polyphylla; mitochondrial apoptosis; reactive oxygen species; saponin.

Figure 1

Low concentration of PPII inhibited glioma cell proliferation. (A) Structure of PPII. (B) PPII inhibited proliferation of U87 and U251 cells in a dose-dependent manner (n=4, 24 h, *, P<0.05; **, P<0.01; ***, P<0.001 compared with normal glioma cells). (C) IC₅₀ value of 3.695 µg/mL PPII for U87 cells and IC₅₀ value of 10.04 µg/mL PPII for U251 cells (n=4, 24 h); (D) iCELLigence showed PPII inhibited proliferation of U87 and U251 cells in a dose- and time-dependent manner (n=3, *, P<0.05 compared with normal glioma cells). PPII, polyphyllin II.

Figure 2

PPII attenuated cell clone and aggression of glioma cells. (A) PPII (Polyphyllin II) reduced the cell clone formation of U87 and U251 cells in a dose-dependent manner (n=3, 14 days, stained with 0.1% crystal violet); (B) effects of PPII attenuating migration and invasion of U87 and U251 cells (n=3, 24 h, stained with 0.1% crystal violet). PPII, polyphyllin II.

Figure 3

PPII induced apoptosis to U87 and U251 cells. (A) Both of high (3.7 µg/mL) and low doses (1.85 µg/mL) of PPII promoted the apoptosis to U87 cells, and both of high (10 µg/mL) and low doses (5 µg/mL) of PPII promoted the apoptosis to U251 cells examined by flow cytometry (n=3, 24 h); (B) TEM revealed that, normal glioma cells exhibited intact cell membranes and normal nuclei, while treated with PPII majority of cells had apoptosis features that mainly include the cytoplasmic shrinkage, the dilation of the endoplasmic reticulum, turgidity of the mitochondrion, the disarrangement, diminution and vacuolization (pointed by arrows, 24 h). PPII, polyphyllin II; TEM, transmission electron microscope.

Figure 4

PPII induced mitochondrial apoptosis in U87 and U251 cells. (A) Western blot results showed 1.85 µg/mL PPII increased Bax, cyt-c, caspase 3 and cleaved caspase 3, and decreased AKT, p-AKT, and Bcl-2 in U87 cells, the protein level was corrected using GAPDH (n=3, “+” represents positive, and “−” represents negative); (B) Western blot results showed 5 µg/mL PPII increased Bax, cyt-c, caspase 3 and cleaved caspase 3, and decreased AKT, p-AKT, and Bcl-2 in U251 cells, the protein level was corrected using GAPDH (n=3, “+” represents positive, and “−” represents negative). PPII, polyphyllin II; Bax, BCL2-associated X protein; cyt-c, cytochrome c; AKT, protein kinase B; p-AKT, phosphorylation protein kinase B; Bcl-2, B-cell lymphoma 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 5

ROS-mediated activation of Bax and cyt-c led to inhibition in PPII-treated U87 and U251 cells. (A) CCK-8 and western blot determined that 4 mg/mL NAC was the optimal dose that could reverse the PPII inhibition via Bax and cyt-c in U87 cells, the protein level was corrected using GAPDH (n=3, ***, P<0.001 compared with PPII treated U87 cell, ○ means not added, ● means added, “+” represents positive, and “−” represents negative); (B) CCK-8 and western blot assay determined that 8 mg/mL NAC was the optimal dose that could reverse the PPII inhibition via Bax and cyt-c in U251 cells, the protein level was corrected using GAPDH (n=3, ***, P<0.001 compared with PPII treated U251 cell, ○ means not added, ● means added, “+” represents positive, and “−” represents negative). PPII, polyphyllin II; CCK-8, Cell Counting Kit-8; NAC, N-acetyl-L-cysteine; Bax, BCL2-associated X protein; cyt-c, cytochrome c; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 6

PPII induced apoptosis in U87 and U251 via ROS-mediated activation of Bax and cyt-c. PPII, polyphyllin II; Bax, BCL2-associated X protein; cyt-c, cytochrome c.