Abstract
Colorectal cancer (CRC) recurrence is often attributable to circulating tumor cells and/or cancer stem cells (CSCs) that resist to conventional therapies and foster tumor progression. Isothiocyanates (ITCs) derived from Brassicaceae vegetables have demonstrated anticancer effects in CRC, however little is known about their effect in CSCs and tumor initiation properties. Here we examined the effect of ITCs-enriched Brassicaceae extracts derived from watercress and broccoli in cell proliferation, CSC phenotype and metastasis using a previously developed three-dimensional HT29 cell model with CSC-like traits. Both extracts were phytochemically characterized and their antiproliferative effect in HT29 monolayers was explored. Next, we performed cell proliferation assays and flow cytometry analysis in HT29 spheroids treated with watercress and broccoli extracts and respective main ITCs, phenethyl isothiocyanate (PEITC) and sulforaphane (SFN). Soft agar assays and relative quantitative expression analysis of stemness markers and Wnt/β-catenin signaling players were performed to evaluate the effect of these phytochemicals in stemness and metastasis. Our results showed that both Brassicaceae extracts and ITCs exert antiproliferative effects in HT29 spheroids, arresting cell cycle at G2/M, possibly due to ITC-induced DNA damage. Colony formation and expression of LGR5 and CD133 cancer stemness markers were significantly reduced. Only watercress extract and PEITC decreased ALDH1 activity in a dose-dependent manner, as well as β-catenin expression. Our research provides new insights on CRC therapy using ITC-enriched Brassicaceae extracts, specially watercress extract, to target CSCs and circulating tumor cells by impairing cell proliferation, ALDH1-mediated chemo-resistance, anoikis evasion, self-renewal and metastatic potential.
Highlights
Colorectal cancer (CRC) is one of the leading causes of mortality worldwide and its incidence is estimated to increase due to the increasing life expectancy of the world’s population and acquisition of Nutrients 2017, 9, 368; doi:10.3390/nu9040368 www.mdpi.com/journal/nutrientsNutrients 2017, 9, 368 unhealthy western lifestyle habits [1]
Cells incubated only with culture medium and cells incubated with the maximum % (v/v) of solvent used were considered as controls–for phenethyl isothiocyanate (PEITC) and watercress and broccoli extracts a maximum of 2.0%, 2.7% and 2.9% of ethanol (v/v) was used, respectively, whereas for SFN it was used a maximum of 1.0% of dimethyl sulfoxide (DMSO) (v/v), to ensure that at the maximum concentrations tested the cytotoxic effects were only dependent on phytochemicals rather than the content of solvent
We demonstrated the applicability of supercritical fluid extraction (SFE) process to isolate PEITC-enriched fractions from watercress
Summary
Colorectal cancer (CRC) is one of the leading causes of mortality worldwide and its incidence is estimated to increase due to the increasing life expectancy of the world’s population and acquisition of Nutrients 2017, 9, 368; doi:10.3390/nu9040368 www.mdpi.com/journal/nutrients. Naturally-occurring dietary compounds have been drawing attention as an appealing strategy to target CRC, including the CSC niche, self-renewal and metabolism [13] In this context, the intake of cruciferous vegetables of the Brassicaceae family (e.g., broccoli and watercress) has been associated to a reduced risk of CRC, due to their high content of glucosinolates [14,15]. Watercress and broccoli were tested in a CRC 3D-cell model derived from HT29 cells and their effect on cell proliferation, cell cycle arrest and targeting of the metastatic and CSC-like phenotype (self-renewal/tumorigenic potential, Wnt/β-catenin signaling, anoikis evasion and drug resistance) was evaluated and compared with the major ITCs, namely PEITC and SFN. The 3D-cell model was previously developed by our group [32], and due to its 3D architecture and establishment of pathophysiological gradients it offers a functional/biological response to treatment that best recapitulates the in vivo tumor behavior and chemotherapeutic response [33]
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