Saikosaponin D inhibits proliferation and induces apoptosis of non-small cell lung cancer cells by inhibiting the STAT3 pathway

Abstract

Objective: To study the effects of saikosaponin D (SSD) on proliferation and apoptosis in human non-small cell lung cancer cell lines, and to explore underlying mechanisms.

Methods: Following treatment with saikosaponin D, A549 and H1299 cells were assessed for anti-proliferation effects using cell cycle kit-8 assays, changes in nuclear morphology using 4',6-diamidino-2-phenylindole (DAPI) staining, and cell apoptosis using annexin V/propidium iodide double staining. Proliferation- and apoptosis-related proteins were detected by immunoblotting.

Results: Saikosaponin D had dose-dependent inhibitory effects on A549 cells (IC₅₀, 3.57 µM) and H1299 cells (IC₅₀, 8.46 µM). DAPI staining revealed decreased cell numbers, and most H1299 cells became round after treatment with 20 µM saikosaponin D. As saikosaponin D concentration increased, the proportions of cells in G0/G1 phase, and cells undergoing apoptosis, increased. Levels of phosphorylated p44/42 and signal transducer and activator of transcription (STAT)3 were significantly downregulated in both cell lines, while total STAT3 levels were not significantly affected. The cleaved form of caspase 3 was significantly upregulated.

Conclusions: Saikosaponin D inhibits proliferation, inducing cell cycle arrest and apoptosis, in lung cancer cells in a dose-dependent manner, possibly through inhibition of STAT3 phosphorylation and activation of caspase 3.

Keywords: STAT3 pathway; Saikosaponin D; apoptosis; lung cancer.

Figure 1.

(a) Diagram showing the chemical structure of saikosaponin D (SSD); and (b) The effects of 24-h treatment with varying concentrations of SSD (0, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, and 25 μM) on inhibition of in vitro proliferation of two non-small cell lung cancer cell lines (A549 and H1299). Data presented as mean ± SD inhibition rate.

Figure 2.

Representative images of phase contrast and DAPI stained cells showing the effect of 24-h treatment with different concentrations of saikosaponin D (SSD), ranging from 0 to 20 μM, on the nucleus of A549 and H1299 cells. Scale bar = 75 μm.

Figure 3.

Effects of 24-h treatment with different concentrations of saikosaponin D (SSD), ranging from 0 to 20 μM, on the cell cycle of A549 and H1299 cells: (a) Representative flow cytometry images of cells in different cell cycle phases; (b) Histogram of flow cytometry results showing proportions of cells in different cell cycle phases; (c) Representative Western blots showing relative levels of the G0/G1 phase proteins Cyclin D1, CDK4 and P27, and β-actin internal controls; and (d) Histogram of Western blot results showing relative protein levels normalised to β-actin (data presented as mean ± SD). *P < 0.05 versus cells treated with 0 µM SSD (controls).

Figure 4.

Effects of 24-h treatment with different concentrations of saikosaponin D (SSD), ranging from 0 to 20 µM, on apoptosis in A549 and H1299 cells: (a) Representative flow cytometry apoptosis profiles in cells treated with 0, 5, 10 or 20 µM SSD for 24 h (R5 quadrant, early apoptotic cells; R3 quadrant, late apoptotic cells); and (b) Distribution of cells in normal phase, or in early or late apoptosis (data presented as mean ± SD). *P < 0.05 versus cells treated with 0 µM SSD (controls).

Figure 5.

Effects of 24-h treatment with different concentrations of saikosaponin D (SSD), ranging from 0 to 20 µM, on the expression of proliferation-related pathway proteins in A549 and H1299 cells: (a) Representative Western blots showing phosphorylated (p)-signal transducer and activator of transcription (STAT)3, STAT3, p-p44/42 and cleaved (cl)-Caspase-3 levels with β-actin internal control; and (b) Semi-quantification of Western blot results showing relative protein levels normalised to β-actin (data presented as mean ± SD). *P < 0.05 versus cells treated with 0 µM SSD (controls).