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Abstract- 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.

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Investigation of factors affecting endothelial cell proliferation is an essential part of angiogenesis studies. Given the importance of inhibiting angiogenesis in the treatment of cancers, and due to the side effects and high cost of anti-angiogenic drugs such as Avastin, the use of physical agents to aid in treatment and reduce the need for high doses of the drug is noteworthy. Magnetic fields are of interest due to their long-distance and non-invasive effects, and many studies have been conducted on their effects on biological phenomena, including angiogenesis, with inconsistent results. In the present study, the effect of a 2 mT alternating magnetic field with a frequency of 200 Hz and Austin on the proliferation of human umbilical vein endothelial cells (HUVEC) was investigated. Cells were treated for 48 hours under a mixture of 50 μg/ml solution of vascular endothelial growth factor (VEGEF) and Avastin at concentrations (zero (drug control), 50, 100, 200 and 400 μg/ml) as well as field treatment groups for They were exposed to magnetic fields for 3, 6, 12, 24 and 48 hours. Then, cell proliferation was assessed using Alamar Blue colorimetric test. Data were analyzed by three-way analysis of variance. According to the findings, the exposure times of 12, 24 and 48 hours showed a significant reduction in cell proliferation compared to the control group, but this difference was not significant in the 3 and 6 hour treatments. Also, the degree of interaction of these factors with each other on HUVEC proliferation was investigated.