The mechanism of Astragalus membranaceus' effects on the proliferation of human basal-like breast cancer cell line MDA-MB-468 from p53/MDM2 pathway.

Abstract

Abstract Abstract #2116 Background: Based on the expression of ER,PR and Her2, breast cancer is classified into several subtypes. Thereinto, basal-like type ("triple-negative" phenotype) is associated with poor prognosis. Patients with this type are unlikely to benefit from currently available targeted therapy. And some researches revealed traditional Chinese medicine could play an important role in breast cancer treatment. p53 mutations can be found in most of basal-like breast cancer patients and this type's cell lines. In our study, we mainly focused on the mechanism of Astragalus membranaceus' effect on the proliferation of MDA-MB-468 cell from p53/MDM2 pathway.
 Material and Methods: The MDA-MB-468 cells were intervened by Astragalus membranaceus Injection (AMI) which contains 1g/ml crude drugs. The effect of AMI on the proliferation of the MDA-MB-468 cells was detected by MTT assay and its time-effect relationship was observed by cell counting kit-8 (CCK-8) assay. At two days after AMI intervention, the mRNA expression of some indicators were assessed by Real-time Quantitative PCR, including p53, MDM2, EGFR, PIP, PI3K, Akt1, Akt2 and PTEN. The anti-proliferation effect of AMI on the cells proliferation was detected by MTT assay after PTEN gene was knocked down by the small interfering RNA (SiRNA), and the protein expressions of EGFR, p-Akt, MDM2 and p53 were obtained by in-cell western assay.
 Results: According to MTT assay, AMI could obviously inhibit the proliferation of the MDA-MB-468 cells (p<0.05) and the effect was stronger when combined with Tarceva (p<0.01); When the PTEN gene was knocked down, AMI only combined with Tarceva could inhibit the cells' proliferation (p<0.005). CCK-8 assay showed that the anti-proliferation effect of AMI was positively correlated with the intervention duration. Real-time Quantitative PCR revealed that AMI could up-regulate the PTEN gene expression (p<0.001) and down-regulate the p53 (p<0.01), MDM2 (p<0.01), Akt2 (p<0.001) and PIP (p<0.001) gene expressions. In-cell western assay indicated that p53 protein was down-regulated at 15min(p<0.05) and 1h(p<0.001), and p-Akt protein expression showed significant down-regulation at 15min(p<0.01), 30min(p<0.001) and 2d(p<0.001) after AMI intervene; when AMI combined with Tarceva, the duration of the down-regulation effect to p53 and p-Akt were all prolonged, and EGFR and MDM2 expressions were down-regulated(p<0.05, p<0.005). After PTEN SiRNA intervene, AMI down-regulate p53 (p<0.001), p-Akt (p<0.005) and EGFR (p<0.05) protein expressions; and its down-regulation effect to p-Akt reduced, but increased to p53, and EGFR.
 Discussion: AMI can significantly inhibit the proliferation of MDA-MB-468 cells and present a time-dependent manner. The effect became stronger after combined with Tarceva. The main mechanism of its anti-proliferation effect may be to activate the positive feedback loop of the p53/ MDM2 pathway. It negatively regulates the PI3K by up-regulating the PTEN gene expression to promote the phosphorylation at D3 point of PIP3, so Akt's phosphorylation level is down-regulated and the cells proliferation was inhibited. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2116.