Plumbagin inhibits cell growth and potentiates apoptosis in human gastric cancer cells in vitro through the NF-κB signaling pathway

Abstract

Aim: To investigate the effects and underlying mechanisms of plumbagin, a naphthoquinone derived from medicinal plant Plumbago zeylanica, on human gastric cancer (GC) cells.

Methods: Human gastric cancer cell lines SGC-7901, MKN-28, and AGS were used. The cell viability was examined using CCK-8 viability assay. Cell proliferation rate was determined using both clonogenic assay and EdU incorporation assay. Apoptosis was detected via Annexin V/propidium iodide double-labeled flow cytometry. Western blotting was used to assess the expression of both NF-κB-regulated gene products and TNF-α-induced activation of p65, IκBα, and IKK. The intracellular location of NF-κB p65 was detected using confocal microscopy.

Results: Plumbagin (2.5-40 μmol/L) concentration-dependently reduced the viability of the GC cells. The IC(50) value of plumbagin in SGC-7901, MKN-28, and AGS cells was 19.12, 13.64, and 10.12 μmol/L, respectively. The compound (5-20 μmol/L) concentration-dependently induced apoptosis of SGC-7901 cells, and potentiated the sensitivity of SGC-7901 cells to chemotherapeutic agents TNF-αand cisplatin. The compound (10 μmol/L) downregulated the expression of NF-κB-regulated gene products, including IAP1, XIAP, Bcl-2, Bcl-xL, tumor factor (TF), and VEGF. In addition to inhibition of NF-κB p65 nuclear translocation, the compound also suppressed TNF-α-induced phosphorylation of p65 and IKK, and the degradation of IκBα.

Conclusion: Plumbagin inhibits cell growth and potentiates apoptosis in human GC cells through the NF-κB pathway.

Figure 1

Plumbagin decreased viability and inhibited proliferation of GC cells. (A) Cell viability in plumbagin-treated SGC-7901, MKN-28, and AGS cells. The cells were treated with indicated concentrations (0–40 μmol/L) of plumbagin for 24 h. (B–C) Plumbagin inhibited the cellular DNA replication in SGC-7901 cells (magnification×200). Cells were incubated with 5 μmol/L plumbagin for 12 h. The EdU-labeled replicating cells were examined under a fluorescent microscope. The red and blue cells were counted in a blind manner. (D–E) Plumbagin decreased the number of colony-forming cells. Cells were treated with indicated concentrations of plumbagin (0, 5, 10, and 20 μmol/L) for 6 h, and then the medium was replaced by fresh medium. Cells were allowed to grow for 10 d. The formed cell clones were counted in a blind manner. The data shown are the mean from three independent experiments.

Figure 2

Plumbagin enhanced cell apoptosis of GC cells. (A) Plumbagin induced apoptosis of SGC-7901 cells. Cells were incubated with 0, 5, 10, and 20 μmol/L plumbagin for 12 h. The apoptosis was analyzed by Annexin V-FITC/PI double-staining assay. (B) The degree of apoptotic cell death was quantified. Data represented the mean±SD of three individual experiments (^bP<0.05).

Figure 3

Plumbagin suppressed the expression of NF-κB-regulated gene products. (A) Plumbagin decreased the expression of NF-κB-regulated anti-apoptotic proteins. (B) Plumbagin suppressed the expression of VEGF and TF. SGC-7901 cells were incubated with 10 μmol/L plumbagin for different time periods (0, 2, 4, 8, 12, and 24 h). Whole-cell extracts were prepared and measured by Western blot analysis using the relevant antibodies.

Figure 4

Plumbagin inhibited TNF-α-induced phosphorylation and nuclear translocation of NF-κB p65. (A) Plumbagin inhibited TNF-α-induced p65 localization by immunofluorescence analysis. SGC-7901 cells were either pretreated or untreated with 5 μmol/L plumbagin for 4 h and then exposed to 0.1 nmol/L TNF-α for 30 min (magnification×400). (B) Plumbagin inhibited TNF-α-induced phosphorylation and nuclear translocation of p65. SGC-7901 cells were first treated or untreated with 5 μmol/L plumbagin for 4 h and then exposed to 0.1 nmol/L TNF-α for indicated times. Nuclear extracts (NE) and cytoplasmic extracts (CE) were prepared and analyzed by Western blot with antibodies against p65 and phospho-p65. The antibodies of anti-β-actin and anti-histone H2B were used as controls.

Figure 5

Plumbagin inhibited TNF-α-induced degradation of IκBα, phosphorylation of IκBα and IKKα. Plumbagin inhibited TNF-α induced IκBα degradation, phosphorylation of IκBα and IKKα. SGC-7901 cells were first exposed to 5 μmol/L plumbagin for 4 h and then treated with 0.1 nmol/L TNF-α for the indicated times and analyzed by Western blotting using various antibodies.

Figure 6

Plumbagin potentiated apoptotic effects of TNF-α and cisplatin. (A–B) SGC-7901 cells were pretreated with 10 μmol/L plumbagin for 2 h, and then treated with 0.1 nmol/L TNF-α and cisplatin for 24 h. Cell viability was then analyzed by the CCK-8 method. Data represented the mean±SD of three individual experiments (^bP<0.05 compared to control group. ^eP<0.05 compared with TNF-α or cisplatin treatment only).