Assessment of raloxifene, estradiol-17β, dl-ormeloxifene and levormeloxifene on thrombin activity.

Abstract Background: Cancer is one of the leading causes of morbidity and mortality globally. Cancer-associated thrombosis is well established in clinical settings, and thrombin has been found to induce angiogenesis at cancer sites. This establishes a link between cardiovascular diseases and cancer, where cancer and thrombin have been intricately associated. Various selective estrogen receptor modulators (SERMs) have been reported to exhibit anticancer activity. Therefore, we investigated estradiol-17β and SERMs dl-ormeloxifene (centchroman), raloxifene and levormeloxifene (l-centchroman) for their anticancer effects and their effect on thrombin activity. Methods: Anticancer activity was assessed against PC-3 cell line by flow cytometry following treatment with estradiol-17β and SERMs at 10 nM-1 mM concentrations. The cells were stained with propidium iodide and the percentage of cells in the sub-G0/G1 region was considered apoptotic. Thrombin inhibitory effect was evaluated by thrombin inhibition assay in vitro following incubation with 100 nM-3 mM concentrations of estradiol-17β or various SERMs. Further, the effect of estradiol-17β and SERMs on endogenous thrombin generation potential (ETP) was assessed by thrombin generation assay on rat plasma in vitro. Results: These compounds exhibited >90% cell death in PC-3 cell lines at 1 mM concentration except estradiol-17β. Neither estradiol-17β, dl-ormeloxifene and levormeloxifene showed any thrombin inhibitory or enhancing activity in thrombin inhibition assay, nor did they show any effect on ETP on rat plasma in vitro. However, raloxifene inhibited thrombin activity in a concentration-dependent manner. Raloxifene decreased ETP of the plasma at 3 and 1 mM,which is equivalent to that of 30-100 U/mL of heparin. Interestingly, raloxifene increased thrombin generation at lower concentrations and it inhibited thrombin generation at higher concentrations. Conclusions: These observations suggest that dl-ormeloxifene, estradiol-17β and levormeloxifene do not possess thrombin inhibitory activity. Raloxifene possesses thrombin modulatory effect in addition to its anticancer activity, and this observation may help us in understanding the thromboembolic complications associated with raloxifene.