Kaempferol Blocks Angiogenesis Through The Suppression Of VEGF-C and HIF-1_ and Prevent Proliferation Through G1 Cell Cycle Arrest In Hepatocellular Carcinoma

Antony Joseph Velanganni AROCKIAM, Bharathidasan University, India
KANNAN M. 1 , UMAPATHI D. 1

1 Molecular Oncology Lab, Dept. of Biochemistry, School of Life Sciences, Bharathidasan University, Tiruchirappalli-24, Tamil Nadu, India

Several pre-clinical studies have shown that Kaempferol has anticancer and anti-inflammatory effect.  However, the exact mechanism of anticancer effect of Kaempferol has not been studied.  Hence, the effect of Kaempferol on cell cycle and angiogenesis in Hepatocellular carcinoma (HCC) has been investigated.  Initially, Kaempferol was tested for its cytotoxicity against Hep3B cells by MTT assay. The effect of Kaempferol on the cell cycle progression was evaluated by flow cytometry. The effects of Kaempferol on the expression of G0/G1 cell cycle regulatory genes were examined. The in vivo model of CAM assay was used to clarify the effect of Kaempferol on liver tumor angiogenesis. Besides, to validate the effect of Kaempferol on tumor angiogenesis, VEGF-C and HIF-1α gene expression were analyzed by RT-PCR. To analyze a possible effect of Kaempferol on migration of Hep3B cell, we analyzed wound closure assays.
Treatment of Hep3B cells with 40 µM (IC50) Kaempferol resulted in the accumulation of cells in the G1 phase when compared to untreated cells. Kaempferol inhibits cell proliferation through G1 cell cycle arrest by activation of P53 gene expression and inhibiting the expression of cyclin D1 and their complex genes of CDK4 and CDK6 in HCC cells. Upon dissection of the CAM of a 12- day old chick embryo, low angiogenic activity in CAM was observed in control. However, high angiogenic activity was found in Hep3B treated.  Moreover, Kaempferol also decreases the expression of angiogenic factors VEGF-C and HIF-1α. The untreated cells closed the wound within 6 hrs, whereas the Kaempferol treated cells does not healed/closed over the same period.  In conclusion, Kaempferol decrease the angiogenesis activity through the suppression of VEGF-C and HIF-1α and prevent proliferation through G1 cell cycle arrest by inhibiting the expression of cyclin D1 and their complex genes of CDK4 and CDK6 in HCC cells.