Effects of bufalin on the proliferation of human lung cancer cells and its molecular mechanisms of action

Abstract

Bufalin, a naturally occurring small-molecule compound from Traditional Chinese Medicine (TCM) Chansu showed inhibitory effects against human prostate, hepatocellular, endometrial and ovarian cancer cells, and leukemia cells. However, whether or not bufalin has inhibitory activity against the proliferation of human non-small cell lung cancer (NSCLC) cells is unclear. The aim of this study is to study the effects of bufalin on the proliferation of NSCLC and its molecular mechanisms of action. The cancer cell proliferation was measured by MTT assay. The apoptosis and cell cycle distribution were analyzed by flow cytometry. The protein expressions and phosphorylation in the cancer cells were detected by Western blot analysis. In the present study, we have demonstrated that bufalin suppressed the proliferation of human NSCLC A549 cell line in time- and dose-dependent manners. Bufalin induced the apoptosis and cell cycle arrest by affecting the protein expressions of Bcl-2/Bax, cytochrome c, caspase-3, PARP, p53, p21WAF1, cyclinD1, and COX-2 in A549 cells. In addition, bufalin reduced the protein levels of receptor expressions and/or phosphorylation of VEGFR1, VEGFR2, EGFR and/or c-Met in A549 cells. Furthermore, bufalin inhibited the protein expressions and phosphorylation of Akt, NF-κB, p44/42 MAPK (ERK1/2) and p38 MAPK in A549 cells. Our results suggest that bufalin inhibits the human lung cancer cell proliferation via VEGFR1/VEGFR2/EGFR/c-Met-Akt/p44/42/p38-NF-κB signaling pathways; bufalin may have a wide therapeutic and/or adjuvant therapeutic application in the treatment of human NSCLC.

Fig. 1

Chemical structure of bufalin (BF)

Fig. 2

Effects of bufalin (BF) on proliferation and induction of apoptosis and cell cycle arrest in A549 cells. A The cells were treated for 48 and 72 h with the indicated concentrations of bufalin (BF1/2.5, BF5/5, BF10/10 μM), LY/50 μM, Bay/10 μM, PD/7 μM, and SB/50 μM. The cells in control group were treated with DMSO [0.1% (v/v), final concentration]. The rate of cell viability was determined by the MTT assay. LY, Bay, PD, and SB are the inhibitors of PI3 K/Akt, NF-κB, p44 (ERK1/2) and p38 MAPK, respectively. B Induction of apoptosis (cells in Sub-G₁ phase) and cell cycle arrest (in G₁ phase) in A549 cells by BF was analyzed by flow cytometry. The cells were treated for 48 h with BF at concentrations of 2.5, 5 and 10 μM. The data are presented as the mean ± SD (Bar) for each group (n = 6). The figures (A, B) are the representative of 3 similar experiments performed. Values with different letters (a–e) differ significantly (p < 0.05)

Fig. 3

Effects of bufalin (BF) on protein expressions of Bcl-2/Bax (A), cytosolic and mitochondrial cytochrome c (cyto c) (B), caspase-3/procaspase-3 (C), and PARP-1 (D) in A549 cells. The cells were treated for 48 h with BF (BF1/2.5, BF5/5, BF10/10 μM), Bay (10 μM) and LY (50 μM). The protein expressions were analyzed by Western Blotting. The optical density (OD) of the band in each panel is normalized to β-actin, respectively. The OD value in each panel is relative to that of control [0.1% (v/v) DMSO vehicle] which is designated as 100%. The average OD value and standard deviation from three assays were calculated. The OD value of the band (normalized to β-actin) in each panel shown as mean ± SD is relative to that of the control designated as 100%. The ratios of each band relative to the control are designated as the ratios (%) of protein expression. LY and Bay are the inhibitors of PI3 K/Akt and NF-κB, respectively. For one experiment, 3 assays were carried out and only one set of gels is shown. Values with different letters (a–e) differ significantly (p < 0.05)

Fig. 4

Effects of bufalin (BF) on protein expressions of p53 (A), p21WAF1 (B), cyclin D1 (C) and COX-2 (D) in A549 cells. The cells were treated for 48 h with BF (BF1/2.5, BF5/5, BF10/10 μM), Bay (10 μM) and LY (50 μM). The protein expressions were analyzed by Western Blotting. The optical density (OD) of the band in each panel is normalized to β-actin, respectively. The OD value in each panel is relative to that of control [0.1% (v/v) DMSO vehicle] which is designated as 100%. The average OD value and standard deviation from three assays were calculated. The OD value of the band (normalized to β-actin) in each panel shown as mean ± SD is relative to that of the control designated as 100%. The ratios of each band relative to the control are designated as the ratios (%) of protein expression. LY and Bay are the inhibitors of PI3 K/Akt, and NF-κB, respectively. For one experiment, 3 assays were carried out and only one set of gels is shown. Values with different letters (a–e) differ significantly (p < 0.05)

Fig. 5

Effects of bufalin (BF) on receptor protein expressions and phosphorylation of pVEGFR2/VEGFR2 (A), VEGFR1 (B), pEGFR/EGFR (C), and p–c-Met/c-Met (D) in A549 cells. The cells were treated for 30 min with BF (BF1/2.5, BF5/5, BF10/10 μM), Bay (10 μM) and LY (50 μM). The protein expressions and phosphorylation were analyzed by Western Blotting. The optical density (OD) of the band in each panel is normalized to β-actin, respectively. The OD value in each panel is relative to that of control [0.1% (v/v) DMSO vehicle] which is designated as 100%. The average OD value and standard deviation from three assays were calculated. The OD value of the band (normalized to β-actin) in each panel shown as mean ± SD is relative to that of the control designated as 100%. The ratios of each band relative to the control are designated as the ratios (%) of protein expression. LY and Bay are the inhibitors of PI3 K/Akt and NF-κB, respectively. For one experiment, 3 assays were carried out and only one set of gels is shown. Values with different letters (a–d) differ significantly (p < 0.05)

Fig. 6

Effects of bufalin (BF) on protein expressions and phosphorylation of pAkt/Akt (A), pp44(42)/p44(42) (B), pp38/p38 (C), and p–c-Met/c-Met (D) in A549 cells. The cells were treated for 30 min with BF (BF1/2.5, BF5/5, BF10/10 μM), Bay (10 μM), LY (50 μM), PD (7 μM) and SB (50 μM). The protein expressions and phosphorylation were analyzed by Western Blotting. The optical density (OD) of the band in each panel is normalized to β-actin, respectively. The OD value in each panel is relative to that of control [0.1% (v/v) DMSO vehicle] which is designated as 100%. The average OD value and standard deviation from three assays were calculated. The OD value of the band (normalized to β-actin) in each panel shown as mean ± SD is relative to that of the control designated as 100%. The ratios of each band relative to the control are designated as the ratios (%) of protein expression. LY, Bay, PD, and SB are the inhibitors of PI3 K/Akt, NF-κB, p44 (ERK1/2), and p38 MAPK, respectively. For one experiment, 3 assays were carried out and only one set of gels is shown. Values with different letters (a–d) differ significantly (p < 0.05)