title: Targeting Colorectal Cancer Proliferation, Stemness and Metastatic Potential Using Brassicaceae Extracts Enriched in Isothiocyanates: A 3D Cell Model-Based Study.
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 fromBrassicaceaevegetables 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-enrichedBrassicaceaeextracts 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 bothBrassicaceaeextracts 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-enrichedBrassicaceaeextracts, 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.
authors.affiliation: iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal. lu.pebre.pereira@gmail.com.|Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), 2780-157 Oeiras, Portugal. lu.pebre.pereira@gmail.com.
authors.forename: Lucília P
authors.initials: LP
authors.lastname: Pereira
authors.affiliation: Unidade de Investigação em Patobiologia Molecular (UIPM), Instituto Português de Oncologia de Lisboa Francisco Gentil, E.P.E (IPOLFG, EPE), 1099-023 Lisboa, Portugal. palsilva@ipolisboa.min-saude.pt.
authors.forename: Patrícia
authors.initials: P
authors.lastname: Silva
authors.affiliation: Unidade de Investigação em Patobiologia Molecular (UIPM), Instituto Português de Oncologia de Lisboa Francisco Gentil, E.P.E (IPOLFG, EPE), 1099-023 Lisboa, Portugal. mrcduarte@ipolisboa.min-saude.pt.
authors.forename: Marlene
authors.initials: M
authors.lastname: Duarte
authors.affiliation: iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal. liliana.rodrigues@ibet.pt.|Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), 2780-157 Oeiras, Portugal. liliana.rodrigues@ibet.pt.
authors.forename: Liliana
authors.initials: L
authors.lastname: Rodrigues
authors.affiliation: iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal. cduarte@itqb.unl.pt.|Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), 2780-157 Oeiras, Portugal. cduarte@itqb.unl.pt.
authors.forename: Catarina M M
authors.initials: CM
authors.lastname: Duarte
authors.affiliation: Unidade de Investigação em Patobiologia Molecular (UIPM), Instituto Português de Oncologia de Lisboa Francisco Gentil, E.P.E (IPOLFG, EPE), 1099-023 Lisboa, Portugal. mc.albuquerque@sapo.pt.
authors.forename: Cristina
authors.initials: C
authors.lastname: Albuquerque
authors.affiliation: iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal. tserra@ibet.pt.|Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), 2780-157 Oeiras, Portugal. tserra@ibet.pt.
authors.forename: Ana Teresa
authors.initials: AT
authors.lastname: Serra
mesh_terms: D016588:Anticarcinogenic Agents / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000972:Antineoplastic Agents, Phytogenic / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D001937:Brassica / Q000737:chemistry* / Q000191:economics; D018938:Caco-2 Cells; D002245:Carbon Dioxide / Q000737:chemistry; D002454:Cell Differentiation; D049109:Cell Proliferation; D015179:Colorectal Neoplasms / Q000178:diet therapy / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D019587:Dietary Supplements / Q000032:analysis; D019008:Drug Resistance, Neoplasm; D015972:Gene Expression Regulation, Neoplastic; D019073:HT29 Cells; D006801:Humans; D017879:Isothiocyanates / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D031223:Nasturtium / Q000737:chemistry*; D009362:Neoplasm Metastasis / Q000473:pathology / Q000517:prevention & control* / Q000628:therapy; D010936:Plant Extracts / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012997:Solvents / Q000737:chemistry; D018874:Spheroids, Cellular; D013454:Sulfoxides
chemical_list: D016588:Anticarcinogenic Agents; D000972:Antineoplastic Agents, Phytogenic; D014408:Biomarkers, Tumor; D017879:Isothiocyanates; D010936:Plant Extracts; D012997:Solvents; D013454:Sulfoxides; D002245:Carbon Dioxide; C058305:phenethyl isothiocyanate; C016766:sulforaphane
keywords: Brassicaceae extracts; broccoli; cancer stem cells; colorectal cancer; metastasis; phenethyl isothiocyanate; sulforaphane; watercress