Abstract
Epithelial to mesenchymal transition (EMT) is a complex biological event, wherein polarized epithelial cells lose their integrity resulting in a mesenchymal phenotype with enhanced motility, a phenomenon known as metastasis. However, the underlying mechanisms of EMT are still poorly understood in cervical carcinomas. In this study, we investigated the molecular signalling events responsible for the effect of TGF-β, a potent inducer of EMT, on HeLa cervical cancer cells. We observed that TGF-β treatment (5 ng/mL) upregulates the expression of EMT-associated transcription factors such as Snail and Slug and downregulates the expression of epithelial markers such as ZO-1 and E-cadherin. Furthermore, treatment with TGF-β activates both Smad-dependent and Smad-independent signaling pathways, which subsides upon addition of Diphenyleneiodonium (DPI), a potent ROS inhibitor that inhibits NADPHoxidase (NOX). TGF-β treatment enhanced cellular migration and invasion ability was diminished in the presence of ROS inhibitors. In addition, we also observed that ROS-mediated, TGF-β-induced EMT progression was inhibited using therapeutic candidates that target the key signal transduction mediators, including PI3K/AKT, ERK, and P38/MAPK. Accordingly, we demonstrated the involvement of redox biology (NOX2 and NOX4 mediate migration and invasion) in TGF-β-mediated EMT advancement and explored suitable therapeutic interventions.
Highlights
Despite several palliative care and preventive measures such as screening, treatment of pre-cancerous and invasive lesions, and vaccination against the causative agent [Human papilloma virus (HPV) 16 and 18], cervical cancer ranks as the fourth most frequent cancer occurring in women worldwide, with an estimated death total of 311,000 women in 2018 alone [5]
In order to study the impact of reactive oxygen species (ROS) on factors relating to the epithelial-mesenchymal transition (EMT), experiments were carried out using 1 h pretreatment with DPI (5 μM) followed by 24 h treatment with transforming growth factor β (TGF-β)
HeLa cells were pretreated for a period of 1 h with inhibitors such as LY294002 (50 μM), U0126 (40 μM), SB203580 (40 μM), SB431542 (10 μM), and PD98050 (20 μM) followed by 24 h treatment with TGF-β to analyze the impact of those inhibitors on proteins relating to Epithelial to mesenchymal transition (EMT) and NADPH oxidase (NOX)
Summary
Despite several palliative care and preventive measures such as screening, treatment of pre-cancerous and invasive lesions, and vaccination against the causative agent [Human papilloma virus (HPV) 16 and 18], cervical cancer ranks as the fourth most frequent cancer occurring in women worldwide, with an estimated death total of 311,000 women in 2018 alone [5]. Reactive oxygen species (ROS) are involved in several biological events related to cancer cell progression, including cell survival, proliferation, invasion, migration, vascular generation, and metastasis; all of which are connected with those cells acquiring EMT phenotypic characteristics [17]. In addition to viral infections, several other compounds such as TGF-β has been shown to induce ROS production and suppress antioxidant system thereby bringing redox imbalance [9]. Despite all these studies, the role of NADPH oxidase (NOX), the major endogenous source of ROS, in cervical cancer progression is not fully elucidated. We used an in vitro approach with HeLa cells and the potent EMT inducer TGF-β to investigate the redox mechanism behind EMT and its associated cellular events that aids in the progression of cervical cancer
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