Simona Dinicola1, Alessia Pasqualato2, Sara Proietti1, Maria Grazia Masiello1, Alessandro Palombo3, Pierpaolo Coluccia4, Rita Canipari5, Angela Catizone5, Giulia Ricci6, Abdel Halim Harrath7, Saleh H Alwasel7, Alessandra Cucina4, Mariano Bizzarri8. 1. Department of Clinical and Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 336, 00161 Rome, Italy; Department of Surgery "Pietro Valdoni", Sapienza University of Rome, Via A. Scarpa 14, 00161 Rome, Italy. 2. Azienda Policlinico Umberto I, viale del Policlinico 155, 00161 Rome, Italy. 3. Department of Surgery "Pietro Valdoni", Sapienza University of Rome, Via A. Scarpa 14, 00161 Rome, Italy; Department of Experimental Medicine, Sapienza University of Rome, Systems Biology Group Lab, viale Regina Elena 324, 00161 Rome, Italy. 4. Department of Surgery "Pietro Valdoni", Sapienza University of Rome, Via A. Scarpa 14, 00161 Rome, Italy; Azienda Policlinico Umberto I, viale del Policlinico 155, 00161 Rome, Italy. 5. Department of Anatomy, Histology, Forensic Medicine and Orthopedics, Sapienza University of Rome, Viale Regina Elena 336, 00161 Rome, Italy. 6. Department of Experimental Medicine, Second University of Naples, Via Santa Maria di Costantinopoli 16, 80138 Naples, Italy. 7. Department of Zoology, College of Science, King Saud University, PO Box 2455, 11451, Riyadh, Saudi Arabia. 8. Department of Experimental Medicine, Sapienza University of Rome, Systems Biology Group Lab, viale Regina Elena 324, 00161 Rome, Italy; Systems Biology Group Lab, Sapienza University of Rome, Rome, Italy. Electronic address: mariano.bizzarri@uniroma1.it.
Abstract
AIM: Presenilin-1 (PS1), the main component of γ-secretase activity support a key role during Epithelial-Mesenchymal Transition (EMT) and chemoresistance acquisition by triggering a complex sequence of molecular events, including E-cadherin down-regulation. However, we hypothesize that EMT and chemoresistance should be deemed separate processes in HCT-8 colon cancer cells. MAIN METHODS: HCT-8 and HCT-8FUres invasion was evaluated by trans-well assay. uPA activity was detected by zymography. Prostaglandin E2 levels were quantified using an ELISA kit. E-cadherin FL and CTF2, PS1, Notch1, Cyclin D1, COX2, SNAI1 and α-SMA expression were determined using Western blot technique. β-Catenin localization was observed by confocal microscopy. Cell apoptosis was evaluated by cytofluorimetric assay, and measurement of caspase-3 and cl-PARP. γ-Secretase activity was inhibited by DAPT, a γ-secretase inhibitor. KEY FINDINGS: Chemoresistant HCT-8 underwent EMT that can be efficiently reversed by inhibiting PS1 activity, leading thus to a normalization of mostly of the pivotal features showed by the invasive cancer phenotype. Indeed, we observed decreased SNAI1 and Notch 1 activation, altogether with reduced E-cadherin cleavage. Concomitantly, resistant HCT-8 invasiveness was almost completely abolished. However, such reversion was not followed by any increase in apoptotic rate, not by changes in E-cadherin levels. Indeed, despite HCT-8FUres underwent an undeniable EMT, full-length E-cadherin levels were found remarkably higher than those observed in wild HCT-8. SIGNIFICANCE: High E-cadherin concentration in presence of enhanced γ-secretase activity is incontestably a paradoxically result, highlighting that E-cadherin loss is not a pre-requisite for EMT. Additionally, EMT and chemoresistance acquisition in HCT-8 should be considered as distinct processes.
AIM: Presenilin-1 (PS1), the main component of γ-secretase activity support a key role during Epithelial-Mesenchymal Transition (EMT) and chemoresistance acquisition by triggering a complex sequence of molecular events, including E-cadherin down-regulation. However, we hypothesize that EMT and chemoresistance should be deemed separate processes in HCT-8 colon cancer cells. MAIN METHODS: HCT-8 and HCT-8FUres invasion was evaluated by trans-well assay. uPA activity was detected by zymography. Prostaglandin E2 levels were quantified using an ELISA kit. E-cadherin FL and CTF2, PS1, Notch1, Cyclin D1, COX2, SNAI1 and α-SMA expression were determined using Western blot technique. β-Catenin localization was observed by confocal microscopy. Cell apoptosis was evaluated by cytofluorimetric assay, and measurement of caspase-3 and cl-PARP. γ-Secretase activity was inhibited by DAPT, a γ-secretase inhibitor. KEY FINDINGS: Chemoresistant HCT-8 underwent EMT that can be efficiently reversed by inhibiting PS1 activity, leading thus to a normalization of mostly of the pivotal features showed by the invasive cancer phenotype. Indeed, we observed decreased SNAI1 and Notch 1 activation, altogether with reduced E-cadherin cleavage. Concomitantly, resistant HCT-8 invasiveness was almost completely abolished. However, such reversion was not followed by any increase in apoptotic rate, not by changes in E-cadherin levels. Indeed, despite HCT-8FUres underwent an undeniable EMT, full-length E-cadherin levels were found remarkably higher than those observed in wild HCT-8. SIGNIFICANCE: High E-cadherin concentration in presence of enhanced γ-secretase activity is incontestably a paradoxically result, highlighting that E-cadherin loss is not a pre-requisite for EMT. Additionally, EMT and chemoresistance acquisition in HCT-8 should be considered as distinct processes.