Baicalin, a Chinese Herbal Medicine, Inhibits the Proliferation and Migration of Human Non-Small Cell Lung Carcinoma (NSCLC) Cells, A549 and H1299, by Activating the SIRT1/AMPK Signaling Pathway

Abstract

BACKGROUND Baicalin is a flavonoid derived from Scutellaria baicalensis, used in Chinese herbal medicine. Activation of the sirtuin 1 gene (SIRT1) and adenosine monophosphate (AMP)-activated protein kinase gene (AMPK), the SIRT1/AMPK signaling pathway, is associated with human malignant tumors. The aim of this study was to investigate the effects of baicalin on the cell viability, apoptosis, proliferation, and migration of human non-small cell lung cancer (NSCLC) cells, A549 and H1299, in vitro. MATERIAL AND METHODS Human NSCLC cells, A549 and H1299, were treated with serial doses of baicalin. Small interfering RNA (siRNA) silencing of the SIRT1 and AMPK genes was performed using cell transfection. The MTT assay was used to determine cell viability, flow cytometry was used to measure cell apoptosis, wound healing and transwell assays were used to assess cell migration of A549 and H1299 cells. Western blotting was used to measure protein expression and phosphorylation levels in untreated A549 and H1299 cells, and cells treated with increasing doses of baicalin. RESULTS Baicalin inhibited the viability, migration, and invasion of A549 and H1299 cells, and increased cell apoptosis in a dose-dependent manner. Baicalin activated the SIRT1/AMPK and mechanistic target of rapamycin (mTOR), and SIRT1/AMPK and matrix metalloproteinase (MMP) signaling in A549 and H1299 cells in a dose-dependent manner. siRNA silencing of SIRT1 and AMPK reduced the effects of baicalin on cell proliferation and migration. CONCLUSIONS Baicalin, a flavonoid used in Chinese herbal medicine, inhibited the proliferation and migration of human NSCLC cells, A549 and H1299, by activating the SIRT1/AMPK signaling pathway.

Figure 1

Effects of baicalin treatment and small interfering RNA (siRNA) silencing of the SIRT1 and AMPK genes on the cell viability and rates of apoptosis of the non-small cell lung cancer (NSCLC) cells, A549 and H1299. The columns on the left of this figure indicate the cell viabilities of cultured A549 cells (white columns) and H1299 cells (black columns) treated with baicalin and/or small interfering RNA (siRNA) silencing of the SIRT1 and AMPK genes, respectively. The columns on the right of this figure include the upper panel that shows the plotted charts of the flow cytometry detection of cell apoptosis; the lower panel show the apoptotic cell rates of cultured A549 cells (white columns) and H1299 cells (black columns) treated with baicalin and/or siRNAs silencing of the SIRT1 and AMPK genes, respectively. * Indicates differences that were statistically significant.

Figure 2

Effects of baicalin treatment on the wound healing assay results of the non-small cell lung cancer (NSCLC) cells, A549 and H1299. The upper panel of the figure shows the fluorescence images of cultured A549 cells and H1299 cells during the wound healing assay. The nuclei of the cells are positively-stained blue with 4′,6-diamidino-2-phenylindole (DAPI). The columns on the lower panel show the percentage of wound closure involving cultured A549 cells (white columns) and H1299 cells (black columns) treated with baicalin and/or small interfering RNA (siRNA) silencing of the SIRT1 and AMPK genes. * Indicates differences that were statistically significant.

Figure 3

Effects of baicalin treatment on cell migration in the transwell assay on the non-small cell lung cancer (NSCLC) cells, A549 and H1299. The upper panel of this figure shows the images of the transwell assay of the cultured cells. Columns on the lower panel indicate the average cell number count per field (total=5 fields) and cells that have penetrated through the membranes of the transwells of cultured A549 cells (white columns) and H1299 cells (black columns) treated with baicalin and/or small interfering RNA (siRNA) silencing of the SIRT1 and AMPK genes. * Indicates differences that were statistically significant.

Figure 4

Effects of baicalin treatment and the Western blotting results on the non-small cell lung cancer (NSCLC) cells, A549 and H1299. The upper part of this figure demonstrates the immunoblots for SIRT1, p-AMPK, AMPK, p-mTOR, mTOR, cleaved caspase-3 (c-caspase-3) encoded by the CASP3 gene, active matrix metalloproteinase (MMP)-2, active MMP-9 and GAPDH in cultured human NSCLC cells, A549 and H1299. The central part (columns) indicates the ratios of SIRT1/GAPDH (white columns), p-AMPK/AMPK (grey columns) and c-caspase-3/GAPDH (black columns) in cultured A549 cells and H1299 cells treated with baicalin and/or small interfering RNA (siRNA) silencing of the SIRT1 and AMPK genes. The columns on the bottom of this figure indicate the ratios of p-mTOR/mTOR (white columns), active MMP-2/GAPDH (grey columns), active MMP-9/GAPDH (black columns) in cultured A549 cells and H1299 cells treated with baicalin and/or small interfering RNA (siRNA) silencing of the SIRT1 and AMPK genes. * Indicates differences that were statistically significant.