Ampelopsin-sodium induces apoptosis in human lung adenocarcinoma cell lines by promoting tubulin polymerization in vitro

Abstract

Previous studies have demonstrated that ampelopsin (AMP), a type of flavonoid isolated from the stems and leaves of Ampelopsis grossedentata, exhibits anti-cancer activity in various types of cancer. Conversion of AMP into its sodium salt (AMP-Na) conferred enhanced solubility and stability to it. The present study aimed to evaluate the anti-cancer activity of AMP-Na in human lung adenocarcinoma cell lines and to investigate its mechanisms of action. Cell proliferation and viability were assessed by MTT and colony formation assays, and cell migration was determined using a scratch wound healing assay. The cell cycle distribution, apoptosis rate and tubulin immunofluorescence intensity were analyzed using flow cytometry, the cell ultra-microstructure was examined using transmission electron microscopy and the accumulation of tubulin was determined using laser confocal microscopy. The results demonstrated that AMP-Na significantly inhibited the proliferation, clonogenicity and migration of human lung adenocarcinoma cells. Furthermore, AMP-Na induced SPC-A-1 cell apoptosis, and promoted tubulin polymerization. The results suggested that the underlying mechanisms of AMP-Na may involve targeting of microtubules and tubulin polymerization to subsequently disrupt mitosis and induce cell cycle arrest at the S-phase.

Keywords: ampelopsin sodium; anti-tumor; apoptosis; human lung adenocarcinoma cell lines; tubulin polymerization.

Figure 1.

AMP-Na decreases lung cancer cell proliferation in vitro. The effect of AMP-Na on (A) SPC-A-1 and (B) A549 cells was assessed using an MTT assay following 48, 72 and 96 h of incubation. The effects of AMP-Na (50 µg/ml) combined with (C) carboplatin, (D) 5-FU and (E) PTX on SPC-A-1 cells were measured by an MTT assay after 48 h of incubation. Three individual experiments were performed. *P<0.05 and **P<0.01 vs. control group; ^#P<0.01 vs. carboplatin, 5-FU or PTX group, respectively; ^∆P<0.01 vs. AMP-Na group. 5-FU, 5-fluorouracil; AMP-Na, ampelopsin-sodium; PTX, paclitaxel.

Figure 2.

Effect of AMP-Na on lung cancer cell colony formation. **P<0.01 vs. control group. AMP-Na, ampelopsin-sodium.

Figure 3.

Effect of AMP-Na on lung cancer cell migration. The effect of AMP-Na on lung cancer cell migration was examined by a wound healing assay. Representative digital images were captured at 0 and 24 h. Magnification, ×10. **P<0.01 vs. control group. AMP-Na, ampelopsin-sodium.

Figure 4.

Effect of AMP-Na on apoptosis and cell cycle arrest in SPC-A-1 cells assessed by flow cytometry. (A) Early and late apoptotic rates and (B) cell cycle distribution in the control and AMP-Na-treated groups. *P<0.05 and **P<0.01 vs. control group. AMP-Na, ampelopsin-sodium; PI, propidium iodide.

Figure 5.

Effect of AMP-Na treatment on the ultra-microstructure of SPC-A-1 cells. Following treatment, cells were fixed and stained with 1% (w/v) uranyl acetate to assess cellular morphology using transmission electron microscopy. (A and B) Cells of the control group and (C and D) cell of the group treated with AMP-Na (100 µg/ml) for 48 h, arrows indicate the morphological characteristics of apoptosis. Scale bars, 5 µm (A and C) or 2 µm (B and D). AMP-Na, ampelopsin-sodium.

Figure 6.

Effect of AMP-Na on microtubulin immunofluorescence. (A) Control cells and cells treated with (B) 12.5, (C) 25 and (D) 50 or (E) 100 µg/ml AMP-Na were fixed, incubated with the primary rat monoclonal tubulin antibody, stained with the FITC-labeled rabbit anti-mouse immunoglobulin G antibody, re-suspended in PBS and analyzed by flow cytometry. A mouse IgG1 isotype control was used for making gates, and the PTX control was used as a positive control (F) MFI of cells treated with AMP-Na at different concentrations. Three individual experiments were performed. **P<0.01 vs. control. AMP-Na, ampelopsin-sodium; MFI, mean fluorescence intensity.

Figure 7.

Effect of AMP-Na on tubulin aggregation. (A) Control cells and cells treated with (B) 25, (C) 50 or (D) 100 µg/ml AMP-Na were fixed, incubated with primary rat monoclonal tubulin antibody, stained with fluorescein isothiocyanate-labeled rabbit anti-mouse immunoglobulin G antibody, re-suspended in PBS and analyzed using a laser confocal microscope (scale bar, 20 µm). AMP-Na, ampelopsin-sodium.