Investigation of apoptosis induction through caspase 9 activation by silibinin in Human Umbilical Vein Endothelial Cells (HUVEC)

Silibinin a natural flavonoid has been reported to induce cell death in various types of cancers and also in endothelial cells which shows its anti-angiogenesis effect. However, its molecular mechanism is not clearly defined. In this article, we provided evidence for one of the mechanisms by which Silibinin induces apoptosis in HUVEC. For this purpose, HUVECs were grown on 96 well plates and cell viability was measured by MTT assay and IC50 was determined as 143μM after 24 hr of treatment by Silibinin. Caspase-9 activity in dose dependent (100-300μM) and time dependent (24,48 and 72hr) treatment by Silibinin was assessed using chromogenic substrate LEHD-pNA. Maximum activity of caspase 9 was in 100 μM of silibinin after 48 hours of treatment. DNA fragmentation was analyzed by gel electrophoresis. Cells were incubated with different concentrations of silibinin (100-400μM) and DNA that was extracted from cells which were incubated by 400 μM of silibinin formed a smear on agarose gel. Data obtained from this study showed the ability of Silibinin to inhibit HUVEC cell proliferation through apoptosis induction which indicates the anti-angiogenesis effect of this compound.