Verbascoside attenuates Rac-1 and HIF-1α signaling cascade in colorectal cancer cells.

OBJECTIVE: Metastasis phenotype is considered as a main challenge in colon cancer therapeutic methods. Furthermore, the side effects of conventional colorectal cancer treatment methods, has attracted a lot of attention into natural ingredients. The aim of the study was to assess the molecular mechanism of verbascoside as natural bio-compound in human HT29 colon cancer cells.

METHODS: HT29 cells were cultured in RPMI-1640 medium containing 10% FBS and 1% penicillin/streptomycin at 37˚C and 5% CO2. HT-29 cells were treated with different concentrations of verbascoside (10, 20, 30, 40, 50, 70, 100 μg/ml) for 24 hours, then MTT assay was used to calculate 50% inhibitory concentration. The migration of the colon cancer cells was evaluated by scratch assay. To evaluate involved anti-proliferative mechanism, Rac-1 (Ras-related C3 botulinum toxin substrate 1) and HIF-1α (hypoxia-inducible factor-1α) related gene expression were evaluated by Real Time PCR.

RESULTS: The results showed that verbascoside inhibited HT29 colon cancer cell proliferation dose-dependently and IC50 was evaluated as 50 μg/ml (***P<0.001). The results of wound healing assay demonstrated verbascoside decreased cell migration in a dose dependent manner. In the IC50 treated HT29 cells metastatic progression was significantly suppressed as **P<0.01. The results of Real Time PCR showed an attenuating effect of verbascoside on Rac-1, Zeb-1 (zinc finger E-box binding homeobox 1), Arp2 (Actin-Related Proteins), Pak1 (p21 (RAC1) activated kinase 1), VEGF (Vascular endothelial growth factor) and HIF-1α as Epithelial-Mesenchymal Transition markers. The down regulation of mRNA levels were Rac-1= 15.38, HIF-1 α = 16.66, Pak-1, Arp-2= 6.25, VEGF=24.39, Zeb-1=35.71 in HT29 cells treated with IC50 concentration of verbascoside.

CONCLUSION: Colorectal cancer cells induce Rac-1 and HIF-1α overexpression which plays an important role in the activation and progression of cell motility, angiogenesis and metastasis. Overall results showed that verbascoside elucidated significant anti-metastatic and anti-invasion activities through suppression of Rac-1, HIF-1α, and Zeb-1 signaling pathway and it may be a suitable candidate to overwhelm colon cancer metastatic phenotype.