Warfarin and coumarin-like Murraya paniculata extract down-regulate EpCAM-mediated cell adhesion: individual components versus mixture for studying botanical metastatic chemopreventives

Cancer metastatic spread is a complex process initiated by the activation, dissemination, seeding and engraftment of circulating tumor cells (CTCs). The series of consequential events include the activation of dormant CTCs, and adhesion between CTCs and vascular endothelial bed of metastatic tissues, and the continued survival and initial proliferation of CTCs after extravasation1,2. We proposed that activation/adhesion of CTCs to the vascular bed is a crucial starting point of the metastatic cascade for chemo-intervention3, and if we can control the starting point, we may effectively prevent cancer metastasis. CTCs are present at low concentrations in the peripheral lymph and blood system of cancer patients and cancer survivors. Many CTCs enrichment and detection methods have been developed, including FISH, immunocytochemistry, RT-PCR, CellSearch system based on the antibody to epithelial cell adhesion molecule (EpCAM; a commonly expressed epithelial cell surface marker4). We recently developed a method of immunomagnetic enrichment coupled with flow cytometry to detect CTCs5, and characterized colorectal CTCs as EpCAM + CD45-pancytokeratin + from the blood of 18 patients. Inspired by the CTC characterization study, we targeted the CTC surface biomarker, in particular the EpCAM, by coating nanomaterial dendrimers with EpCAM antibody to specifically capture blood CTCs and restrain their activity without any cytotoxic effects6,7,8. We also demonstrated that a nitric oxide donor compound could inhibit CTCs-initiated metastasis cascade by directly producing vasorelaxation and interfering with hetero-adhesion of cancer cells to vascular endothelium via down-regulating expression of cell adhesion molecules9. Very recently, we showed that the highly active metastasis prevention therapy (abbreviated as HAMPT, a combination of four old but safe drugs) can effectively prevent cancer metastasis by acting on intervening inflammatory factors, cell adhesion molecules, selectins, integrins, and platelets to prevent CTCs from seeding on extracellular matrices, and strengthening the extracellular matrices3. Recently we reviewed the current literature and analyzed the molecular and cellular similarities and differences between embryonic implantation to uterine endometrium and CTCs adhesion to vascular endothelium, and found that many molecules, including epithelial cell adhesion molecule (EpCAM), intercellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), selectin, integrin, hormones, sialyl lewis X, and mitochondrial membrane potential (MMP), are shared by both the embryonic implantation and cancer cell adhesion-invasion systems10,11,12,13. The analysis led us to screening the traditional abortion Chinese medicinal plants or herbs for potential safe and effective metastatic chemopreventives. In the huge treasure of the Traditional Chinese Medicine (TCM), we hunted for those TCM that should possess the following properties: safe, anti-adhesion, anti-inflammation, anti-coagulation, analgesic, and vasodilation. The TCM Murraya paniculata (L.) Jack meets the above criteria14,15,16. The extract from this TCM appeared to be very safe with the oral LD50 value to mice > 5 g/kg (the maximum mouse intragastric administration volume)17. We first identified the most-promising extracted fraction G (containing flavonoids and coumarins) from the TCM’s raw ethanol/dichloromethane extracts by using the bioactivity-guided fast screen assay18. The G extract showed specific inhibition on both embryonic implantation to human endometrial bed and adhesion of cancer cells to human endothelium in a concentration-dependent manner without significant cytotoxicity19. The inhibition resulted from down-regulation by the G extract on expression of integrin, CD44, and E-selectin. The extract G also inhibited invasion and migration of cancer cells. Oral administration of the extract G to the immunocompetent mice inoculated with mouse melanoma cells produced significant inhibition on lung metastasis without marked side effects. To continue the previous discovery, in the present study, we used the phytochemically separated components from the root of Murraya paniculata (L.) to explore their molecular and cellular bioactivities in preventing adhesion of cancer cells to vascular endothelium after phytochemically characterizing the components (Fig. 1). Since TCM or botanical medicine is often used as an extract that contains various agonistic and antagonistic components (or Ying and Yang components defined by the TCM phrase), and the extract only exhibits a leading pharmacological effect after harmonizing each individual effects, we therefore dissected the anti-adhesion effects of both component mixture (or reconstituting each components together according to their original % proportion in the extract) and individual components by running comparative bioassays in parallel. The studies were reported below. Figure 1 Schematic of bioactivity-guided fast screen for cancer metastatic chemopreventive materials from raw extracts of Murraya paniculata.