Objective: Metastasis phenotype is considered as the main challenge in colon cancer therapeutic
methods. Furthermore, the side effects of conventional colorectal cancer treatment methods have 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 antiproliferative
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 was 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.
Detection of colorectal cancer cells from feces using quantitative real-time RT-PCR for colorectal cancer diagnosis