Primary Study Of The Inhibition Of ACA On CRM1 Dependent Nuclear Export

1'-acetoxychavicol acetates (ACA) is a kind of small molecular chemical compound which is extracted from the galangal. ACA possesses a lot of biological functions, such as anti-cancer, anti-allergy, anti-fungi, protect stomach and so on.We tested the drug effect of ACA in several mammary tumor cell lines, such as ZR-75-30,SK-BR-3,MDA-MB-453,MCF-7,Bcap-37; the results of test showed that ACA possesses the ability to inhibit the normal growth of those tumor cells in a concentration of 10μmol/L. In the meantime, we tested the normal mammary cell line HBL-100, and found that no obvious inhibit effect was observed at the same drug concentration.The GFP signal can track the location of fusion protein GST-NLS-GFP-NES in the Schizosaccharomyces pombe (S.pombe). When the S.pombe incubated with ACA, we found that most of the GFP signal cumulates in the nucleus of S.pombe, which indicated that ACA inhibits CRM1 function and blocks the export of NES protein.According to the inhibition of ACA on CRM1, we formulated the hypothesis that ACA affect the biological function of CRM1 by directly binding to CRM1. Therefore, given that LMB binds to CRM1 and inhibit its function, we performed the competitive assay between ACA and biotinylated LMB. The experiment result demonstrates that high concentration ACA could compete with LMB.In order to elucidate the interaction between ACA and CRM1, we constructed eukaryotic expression vector, pcDNA-CRM1-HA, which expressed the full length of CRM1 protein. We also prepared the prokaryotic expression vectors, pGEX-4T2-CRM1-I(1-1029), pGEX-4T2-CRM1-II(985-2199), pGEX-4T2-CRM1-III(2110-3213), which express the different part of CRM1 protein separately.Furthermore, we use the TNT in vitro translation system to express the CRM1 protein by pcDNA-CRM1-HA. The CRM1 protein was incubate with gradient concentration of ACA. The treated protein samples were run in native poly-acrylamide gel and transferred to a PVDF. The immunoblot results showed that high concentration ACA caused the CRM1 mobility shift, which may caused by the protein structural conformation changes. Thus we conclude that ACA may interact with CRM1 by direct binding.In summary, although more and solider experiment data are needed to confirm the hypothesis the ACA interact with CRM1 by directly binding, our research contribute to the further understanding of the mechanism of ACA which may be important to the novel therapies for cancer.