The anti-invasive activity of synthetic alkaloid ethoxyfagaronine on L1210 leukemia cells is mediated by down-regulation of plasminogen activators and MT1-MMP expression and activity

Abstract

Quaternary benzo[c]phenanthridines such as fagaronine are natural substances which have been reported to exhibit anticancer and anti-leukemic properties. However, the therapeutic use of these molecules is limited due to the high dose required to exhibit anti-tumor activity and subsequent toxicity. In this study, we describe the therapeutic potential of a new derivative of fagaronine, Ethoxyfagaronine (N-methyl-12-ethoxy-2hydroxy-3, 8, 9-trimethoxybenzo[c]-phenanthridiniumchlorhydrate) as an anti-leukemic agent. Cytotoxic activity and cell growth inhibition of Ethoxyfagaronine (Etxfag) was tested on murine L1210 leukemia cells using trypan blue assay and MTT assay. At the concentration of 10(-7) M, Etxfag induced less than 10% of cell death. Etxfag (10(-7) M) was tested on L1210 cell invasiveness using matrigel™ precoated transwell chambers and efficiently reduces the invasive potential of L1210 cells by more than 50% as compared with untreated cells. Western blot and immunofluorescence experiments showed that Etxfag decreased both MT1-MMP expression and activation at the cell surface, decreased plasmin activity by down-regulating u-PAR and uPA expression at the cell surface and increasing PAI-1 secretion in conditioned media. The set of our findings underscore the therapeutic potential of ethoxyfagaronine as a new potential anticancer agent able to prevent leukemic cell dissemination.

Effects of nutrients on matrix metalloproteinases in human T-lymphotropic virus type 1 positive and negative malignant T-lymphocytes

“…In patients suffering from ATL cell infiltration, a significant increase in plasma MMP-9 was observed (26). Moreover, MMP-2 expression has been shown to be correlated with multi-organ extramedullary infiltration in adult acute lymphoblastic leukemia (ALL) (27). These MMPs have been dubbed as potential prognostic and diagnostic biomarkers in various cancer types and stages and have therefore been targeted by a wide variety of inhibitors, only to be met with failure in clinical trials (4).…”

Targeting Focal Adhesion Assembly by Ethoxyfagaronine Prevents Lymphoblastic Cell Adhesion to Fibronectin

“…35 × 10 4 cells/mL were plated on tissue culture plates (25 cm 2 ) coated with FN/V (50 g/mL) or BSA 1% (w/v) and incubated for 2 h at 37 • C. The non-adherent cells were discarded and the plates rinsed thrice with ice-cold PBS containing 50 M Na 3 VO 4 .The adherent cells were lysed in 200 L lysis buffer and cell lysates were analysed as described [22].…”

“…Cells were washed with ice-cold PBS containing 20 M Na 3 VO 4 and incubated with 0.5 mg/mL EZ-Link ™ Sulfo-NHS-LC-Biotin, a membrane impermeable biotinylation reagent, in PBS at 4 • C for 30 min. Cell surface proteins were separated as described previously and analysed by SDS-PAGE followed by western blotting [22].…”

“…Numbers of cycles were adjusted to ensure that amplifications were in a linear range. Real-time PCR was performed as described previously [22] to assess expression of ␤1 integrin genes. The entire experiment from RNA extraction to realtime PCR steps was repeated four times with different sets of samples.…”

“…Ethoxyfagaronine (etxfag), a hemi-synthetic derivative of fagaronine, exhibits a high DNA binding affinity but a low cytotoxicity in vitro [20,21]. We recently reported that etxfag reduces L1210 lymphoblastic cell invasiveness through a down-regulation of plasminogen activators and MT1-MMP expression and activation [22]. We now investigate etxfag effects on L1210 cell adhesion.…”