| Background and ObjectXiaoaiping preparation, an extract of Marsdenia tenacissima (MTE), has inhibitory effects on tumor growth. Lung cancer is the most common cause of cancer-related death worldwide and non-small cell lung cancer (NSCLC) accounts for about 85% of the cases. Tumour suppressor p53 deficiency has been often found in lung cancer. Our previous study observed the biological effects of changed expression of protein lysine arginine methyl transferase enzyme SET7/9 on p53 deficient NSCLC cells, and showed that upregulation of SET7/9 inhibited apoptosis and downregulation of SET7/9 induced apoptosis. This study investigates the effects of MTE on proliferation of p53 deficient NSCLC cells and explores the corresponding mechanisms including whether Set7/9 involved.MethodsNSCLC A549 and NCI-H1299 cells were chosen and divided into MTE-treated and-untreated (control) groups. For MTE-treated groups, the cells were treated with 30.0 mg/mL, 50.0 mg/mL or 80.0 mg/mL of MTE for 24 h,48 h or 72 h, respectively. Meanwhile, the cells in control groups were treated with the same volume of saline. Cell growth and morphology were observed under an inverted microscope. The CCK-8 assay was used to measure the cell viability and proliferation. Hoechst33342 staining and Annein V-FITC/PI double staining were used to detect the apoptosis. Cell cycle distribution was detected by flow cytometry. SET7/9, p65 and p21 levels were detected by Western blot. ResultsCompared with the control, the cell densities of MTE-treated groups of both cell lines were decreased and their morphology changed in 48 h. More NCI-H1299 cells appeared to be suspension but the number of cells with nuclear shrinkage and deep dyeing was not increased when treated with 80.0 mg/mL of MTE. The CCK-8 assay showed that survival cells reduced in MTE-treated groups of these lines in a MTE concentration-or treatment time-dependant manner, The median cell lethal concentration (IC50) for NCI-H1299 cells were 60.0,37.6, 31.0 mg/mL, while the IC50 for A549 cells were 90.1,74.5 and 64.1mg/mL, when these cell lines were treated with MTE for 24,48, or 72 h, respectively. Meanwhile, no significant changes of apoptosis rates were found in the cell lines with treatments of different concentration of MTE for 48 h. Flow cytometry analysis showed that the percentages of S phase were (44.1±0.4)%, (51.7±2.0)% and (59.8±3.8)% in NCI-H1299, while (21.8±4.8)%, (25.5±0.6)% and (32.8±0.9)% in A549 cells with 30.0,50.0 or 80.0 mg/mL of MTE treatment for 48 h, respectively. Compared with that of controls [(42.3±0.8)% of NCI-H1299 cells and (21.5±5.8)% of A549 cells without MTE treatment], the percentages of S phase increased obviously in the cell lines with treatments of different MTE concentration except 30.0 mg/mL. Western blot analysis showed that the levels of both SET7/9 and p65 decreased and that of p21 elevated when the lines treated with increased concentrations of MTE.Conclusions(1) Xiaoaiping prepartion can inhibit the proliferation of p53 deficient NSCLC NCI-H1299 and A549 cells in the manner of treatment concentration or time dependence.(2) Xiaoaiping prepartion suppresses the cell proliferation through cell-cycle arrest but not apoptosis.(3) The intracellular pathways mediated by Set7/9, p65 and/or p21 may be involved in inhibitory effects of Xiaoaiping prepartion on proliferation of these p53 deficient NSCLC cells. |
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