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Journal of Chinese Integrative Medicine ›› 2011, Vol. 9 ›› Issue (12): 1339-1346.doi: 10.3736/jcim20111210

• Original Experimental Research • Previous Articles     Next Articles

Effects of Astragalus polysaccharide on proliferation and Akt phosphorylation of the basal-like breast cancer cell line

Mei-na Ye1, Hong-feng Chen1(), Rui-juan Zhou1, Ming-juan Liao2   

  1. 1. Department of Breast Surgery, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China
    2. Department of Traditional Chinese Medicine, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China
  • Received:2011-06-01 Accepted:2011-08-08 Online:2011-12-20 Published:2011-12-15

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Objective: To investigate the effects of Astragalus polysaccharide (APS) on proliferation of basal-like breast cancer cell line MDA-MB-468 cells and Akt phosphorylation in MDA-MB-468 cells.
Methods: APS at different concentrations was used to culture MDA-MB-468 cells for different time periods, and then proliferation of MDA-MB-468 cells was assayed using methyl thiazolyl tetrazolium (MTT) assay to determine the time- and dose-dependent effects of APS. For observing the effects of APS on phosphor-Akt (p-Akt), in-cell Western blot method was used after 1, 2, 4 and 7 d of culture in APS to detect protein expressions of p-Akt (Thr308) and p-Akt (Ser473). Protein levels of the key targets in p53/murine double minute 2 (MDM2) signaling pathway, such as p53, MDM2 and phosphatase and tensin homolog deleted on chromosome ten (PTEN) were also detected. After PTEN gene was silenced by small interfering RNA (siRNA) in MDA-MB-468 cells, expressions of p-Akt (Thr308 and Ser473) were assayed by the in-cell Western blot method after 2 d of APS treatment.
Results: APS at 1 and 0.5 mg/mL concentrations effectively inhibited the proliferation of MDA-MB-468 cells and was used in subsequent tests. Compared with the control group, APS decreased the protein expression of p-Akt (Thr308) in MDA-MB-468 cells after 1-, 2-, 4- and 7-day culture, and also decreased the protein expression of p-Akt (Ser473) and up-regulated the protein expression of MDM2 in MDA-MB-468 cells after 1- and 2-day culture. Expressions of p53 and PTEN were up-regulated after 7 d of APS culture. After silencing PTEN gene by siRNA, APS could not mediate Akt phosphorylation.
Conclusion: APS can inhibit proliferation of basal-like breast cancer cell line MDA-MB-468, and down-regulate the expression of Akt phosphorylation. The antiproliferation mechanisms may be related to its effects of up-regulating the expressions of p53 and PTEN by regulating p53/MDM2 positive and negative feedback loops.

Key words: astragalan, breast neoplasms, basal-like breast cancer, Akt phosphorylation, MDA-MB-468 cell line, cell proliferation, PTEN phosphohydrolase

Table 1

Effects of APS on proliferation of MDA-MB-468 cells (x±s)"

Group n Concentration (mg/mL) Absorbance value Inhibition rate (%)
Control 6 0 0.362±0.043 0
APS 6 5 0.181±0.024** 50.1
6 2.5 0.191±0.026** 47.3
6 1 0.204±0.023** 43.6
6 0.5 0.270±0.037**△△ 25.4
6 0.25 0.344±0.068△△ 4.9
6 0.1 0.368±0.019△△ -1.7

Table 2

Time-dependent effects of APS on proliferation of MDA-MB-468 cells (x±s)"

Time (d) n Control APS (1 mg/mL) APS (0.5 mg/mL)
Absorbance value Inhibition
rate (%)		 Absorbance value Inhibition
rate (%)		 Absorbance value Inhibition
rate (%)
0 6 0.169±0.019 0 0.167±0.016 1.2 0.162±0.010 4.1
1 6 0.174±0.006 0 0.152±0.012 12.6 0.142±0.009** 18.4
2 6 0.237±0.008 0 0.134±0.008** 43.5 0.170±0.010**△△ 28.3
3 6 0.302±0.014 0 0.227±0.007** 24.8 0.239±0.009**△ 20.9
4 6 0.349±0.016 0 0.179±0.015** 48.7 0.191±0.012** 45.3
5 6 0.381±0.035 0 0.212±0.015** 44.4 0.228±0.017** 40.2
6 6 0.457±0.039 0 0.278±0.033** 39.2 0.253±0.013** 44.6
7 6 0.469±0.025 0 0.241±0.014** 48.6 0.268±0.010**△△ 42.9

Table 3

Effects of APS on expressions of p-Akt (Thr308), p-Akt (Ser473), p53, PTEN and MDM2 (x±s)"

Group n p-Akt (Thr308) p-Akt (Ser473) p53 PTEN MDM2
Control
1 d 3 1.251±0.100 0.268±0.002 0.996±0.034 0.160±0.006 0.773±0.031
2 d 3 1.550±0.036 0.260±0.016 0.861±0.041 0.147±0.001 0.741±0.034
4 d 3 0.596±0.059 0.111±0.012 1.407±0.224 0.288±0.023 0.227±0.026
7 d 3 1.214±0.059 0.256±0.015 1.461±0.036 0.169±0.042 0.238±0.033
APS (1 mg/mL)
1 d 3 1.057±0.035* 0.230±0.004** 0.925±0.043 0.159±0.008 0.988±0.045**
2 d 3 1.139±0.083** 0.211±0.013** 0.910±0.067 0.151±0.005 0.995±0.052**
4 d 3 0.488±0.025** 0.101±0.014 1.205±0.113* 0.256±0.006* 0.203±0.017
7 d 3 0.850±0.083** 0.210±0.006 1.752±0.080** 0.208±0.011* 0.239±0.024
APS (0.5 mg/mL)
1 d 3 1.059±0.068* 0.229±0.004** 0.885±0.023* 0.151±0.002 0.922±0.047**
2 d 3 1.111±0.073** 0.161±0.005** 0.919±0.145 0.145±0.005 1.101±0.107**
4 d 3 0.470±0.042** 0.105±0.003 1.122±0.053** 0.257±0.003* 0.211±0.033
7 d 3 0.835±0.073** 0.207±0.013 1.707±0.155** 0.210±0.015* 0.206±0.041

Table 4

Effects of APS on proliferation of MDA-MB-468 cells after silencing of PTEN gene by siRNA (x±s)"

Group n Non-target siRNA-treated PTEN on-target siRNA-treated
Absorbance value Inhibition rate (%) Absorbance value Inhibition rate (%)
Control 3 0.237±0.008 0 0.539±0.027 0
APS (1 mg/mL) 3 0.134±0.008** 43.46 0.408±0.038** 24.30
APS (0.5 mg/mL) 3 0.170±0.010** 28.27 0.423±0.021** 21.52

Table 5

Effects of APS on expression of p-Akt (Thr308) after silencing of PTEN gene by siRNA (x±s)"

Group n Ratio of fluorescence value
Non-target siRNA-treated PTEN on-target siRNA-treated
Control 3 1.550±0.036 1.457±0.167
APS (1 mg/mL) 3 1.139±0.083** 1.355±0.106
APS (0.5 mg/mL) 3 1.111±0.073** 1.368±0.010

Table 6

Effects of APS on expression of p-Akt (Ser473) after silencing of PTEN gene by siRNA (x±s)"

Group n Ratio of fluorescence value
Non-target siRNA-treated PTEN on-target siRNA-treated
Control 3 0.260±0.016 0.567±0.024
APS (1 mg/mL) 3 0.211±0.013** 0.521±0.029
APS (0.5 mg/mL) 3 0.161±0.005** 0.511±0.046

Figure 1

Positive and negative feedback loops of p53/MDM2 pathway PI3K: phosphatidylinositol 3-kinases; PIP3: phosphatidylinositol 3,4,5-triphosphate; Akt: protein kinase B; MDM2: murine double minute 2; PTEN: phosphatase and tensin homolog deleted on chromosome ten."

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