Anti-metastatic Effect Of Ionone Alkaloids From Pachysandra Terminalis And Its Mechanism

Objective:To screen the anti-metastatic activity of ionone alkaloids on human breast cancer cell line MDA-MB-231by chemotaxis assay and explore its mechanism, so as to find new potential targets and anti-metastatic lead compounds.Methods:1. Chemotaxis assay was utilized to detect the effect of ionone alkaloids (compound1,2, and3) from Pachysandra terminalis Sieb.Zucc on human breast cancer cell line MDA-MB-231chemotaxis activity. Further more, we studied the anti-metastatic effect in vitro of compound1which showed significantly decreased chemotaxis ability. Wound healing assay and Transwell assay were used to examine anti-migration activity of compound1on MDA-MB-231and HUVEC in vitro. The tube formation assay of HUVEC was used to study anti-angiogenic effect of compound1.2. Western blot detected the phosphorylation level of tumor metastasis-associated signaling proteins (Akt, PKCζ, and integrinβ1) in MDA-MB-231cells treated with different concentrations of compound1.3. In order to explore the structure-activity relationship of ionone alkaloids, we have synthesized a series of ionone alkaloid derivatives, and evaluated their anti-metastatic activity by chemotaxis assay.Results:1. Compound1can significantly reduce chemotaxis ability of human breast cancer cell line MDA-MB-231in a dose-dependent manner. At a concentration of10μM, the inhibition rate reached78.6%, and the IC50value was1.49μM. Compound1clearly revealed migration inhibitory activity on breast cancer cells MDA-MB-231and HUVEC in vitro (P＜0.05). Compound1also inhibited capillary-like tube formation of HUVEC in a dose-dependent manner.2. Compound1reduced the phosphorylation level of Akt, PKCζ and integrinβ1in a dose-dependent manner.3. Compound5,6,7,8,13,18,22and23significantly inhibited the number of migrated cells through the polycarbonate membrane at a concentration of25μM, the inhibition rate reached71.4%,58.7%,58.0%,61.5%,75.0%,74.2%,41.9%and43.2%respectively.Conclusion:1. Compound1significantly inhibited chemotaxis ability, migration and invasion ability of human breast cancer cell line MDA-MB-231, which showed its specific anti-metastatic effect. Compound1inhibited the migration of HUVEC in vitro, and inhibited the capillary-like tube formation, which suggested compound1had a potential anti-angiogenic effect.2. Compound1reduced the phosphorylation levels of Akt, PKCζ and integrinβ1. The anti-metastatic effect of compound1on MDA-MB-231cells might be attributed to down-regulate the phosphorylation levels of Akt, PKCζ and integrinβ1.3. About the structure-activity relationship analysis of ionone alkaloid, we believe that the action loci space of ionone alkaloids may be relative limited, and7,8-double bond is a necessary. Further more, benzyl substituted compounds with electron-donating group showed better anti-metastatic activity than electron-withdrawing group.