Regulation of MAPKs Signaling Contributes to the Growth Inhibition of 1,7-Dihydroxy-3,4-dimethoxyxanthone on Multidrug Resistance A549/Taxol Cells

Abstract

1,7-Dihydroxy-3,4-dimethoxyxanthone (XAN) is a bioactive compound isolated from Securidaca inappendiculata Hassk. and validated with antiproliferative activities on a panel of cancer cell lines. This study was designed to investigate its growth inhibitory effects on multidrug resistance (MDR) non-small cell lung carcinoma (NSCLC) cell line A549/Taxol and explore the possible linkage between modulation of MAPKs and the bioactivities. Its growth inhibitory potency on the cells was estimated by MTT assay, and flow cytometric analysis was employed to investigate its potential cell cycle arrest and proapoptosis effects. Expressions of hallmark proteins were assessed by Western-Blot method. The results showed A549/Taxol cells were sensitive to XAN. XAN inhibited the proliferation of A549/Taxol cells in the time and concentration dependent manners. It acted as a potent inducer of apoptosis and cell cycle arrest in the cells. Western-Blot investigation validated the proapoptosis and cell cycle arrest activities of XAN and the potential of MDR reversion. Upregulation of p38 by XAN, which accounted for the cell cycle arrest at G2 phase, and the downregulation of ERK associated with the proapoptosis activity were also revealed. Further analysis found p53 may be the central role mediated the bioactivities of MAPKs in A549/Taxol cells. Based on these evidences, a conclusion has been deduced that XAN could be a potential agent for MDR NSCLC therapy targeting specifically MAPKs.

Figure 1

XAN inhibited the proliferation of A549/Taxol (A549) cells in vitro. Results were assessed by MTT assay; values are presented as mean ± SD, n = 3.

Figure 2

Morphology of A549/Taxol cells. (a) Normal; (b) XAN (5.2 μM); (c) XAN (20.8 μM); (d) XAN (20.8 μM) + SB (25 μM); (e) XAN (20.8 μM) + U0126 (10 μM); (f) XAN (20.8 μM) + SP (25 μM).

Figure 3

XAN induced apoptosis of A549/Taxol cells, and MAPKs inhibitors exerted divergent effects on the results. (a) Normal; (b) XAN (5.2 μM); (c) XAN (20.8 μM); (d) XAN (20.8 μM) + SB (25 μM); (e) XAN (20.8 μM) + SP (25 μM); (f) XAN (20.8 μM) + U0126 (10 μM).

Figure 4

XAN induced cell cycle arrest of A549/Taxol cells at G2 phase, and SB reversed these effects. (a) Normal; (b) XAN (5.2 μM); (c) XAN (20.8 μM); (d) XAN (20.8 μM) + SB (25 μM). ^∗ P < 0.05, ^∗∗ P < 0.01 compared with normal control; ^# P < 0.05 compared with XAN (20.8 μM).

Figure 5

XAN (and inhibitors) modulated MAPKs and main downstream signalings in A549/Taxol cells. (a) 8 h after treatment; (b) 36 h after treatment. Unit of concentration of XAN and inhibitors was μM. ^∗ P < 0.05, ^∗∗ P < 0.01 compared with normal control; ^# P < 0.05, ^## P < 0.01 compared with XAN (20.8 μM).

Figure 6

XAN (and inhibitors) modulated MAPKs in A549 cells after the 36 h treatment. Unit of concentration of XAN and inhibitors was μM. ^∗ P < 0.05, ^∗∗ P < 0.01 compared with normal control; ^# P < 0.05, ^## P < 0.01 compared with XAN (20.8 μM).