Role of NAPDH oxidase and its therapeutic intervention in TGF-β-mediated EMT progression: an in vitro analysis on HeLa cervical cancer cells
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 NAPDH oxidase (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.
KeywordsCervical cancer EMT ROS TGF-β NAPDH oxidase
extra cellular matrix
epithelial to mesenchymal transition
human papilloma virus
reactive oxygen species
transforming growth factor β
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 . Although numerous studies have been carried out to understand the complex pathways involved in the progression of cervical cancer cells, a complete understanding of how this cancer advances is still not available . In order to develop therapeutic candidates to treat cervical cancer, we must first understand the underlying biomolecular signalling mechanisms that induce cancer progression.
Epithelial to mesenchymal transition (EMT) is a complex process in which polarized epithelial cells undergo numerous biological changes, such as spindle-shaped cellular morphology, dissociation of cellular tight junctions, upregulation/downregulation of mesenchymal/epithelial markers, excessive production of extracellular matrix components, and induction of EMT-associated transcription factors, that result in the acquisition of mesenchymal characteristics [4, 14]. In the context of physiological tissues, the process of EMT is typically divided into three stages. Stage I occurs during embryogenesis and organ development, Stage II occurs during tissue regeneration and the healing process, and Stage III occurs during cancer advancement and metastasis. The phenotypic changes, as well as the invasive and metastatic progression of cancer cells increases drastically during Stage III of EMT . Recent findings support that several EMT-associated regulatory factors are redox sensitive, and thus, explicating the EMT mechanism in the context of redox biology is valuable for exploring the key molecular events underlying cancer progression .
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 also connected with those cells acquiring EMT phenotypic characteristics . Several endogenous (altered P450 metabolism, activation of inflammatory cells, oxidative phosphorylation etc.) and exogenous factors (viral infections, radiation, chemicals, pollutants etc.) contribute to the production of reactive oxygen/nitrogen species , which ultimately results in oxidative stress induced damages associated with nucleic acid and protein, thereby leading to cancer malignancy. In cervical cancer, persistent HPV infection and the interaction of HPV oncoprotein components E6 and E7 with tumor suppressor factors and cell cycle regulators lead to the production of ROS followed by cell transformation process . Further numerous antioxidant proteins were found to be deregulated in HPV-associated penile and cervical carcinomas by modulating the accumulation of oxygen species. 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 . Despite all these studies, the role of NAPDH oxidase (NOX), the major endogenous source of ROS, in cervical cancer progression is not fully elucidated. Thus, in this present study, we planned to study the role of ROS contributor—NAPDH oxidase (NOX) and its therapeutic intervention in TGF-β-mediated EMT progression in HeLa cervical cancer cells. For this, 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.
Materials and methods
Cell lines and reagents
The HeLa human cervical cancer cell line (American Type Culture Collection CCL-2), fetal bovine serum (FBS, Gibco Inc., NY, USA), and Dulbecco’s Modified Eagle Medium (DMEM, Corning, NY, USA) were used in this study. The chemicals used in this study included transforming growth factor β1 (TGF-β1) (Invitrogen, CA, USA), diphenyleneiodonium (DPI), apocynin (APO), and N-acetyl cysteine (NAC) (Sigma-Aldrich, MO, USA), GKT137831 (Cayman Chemical, Michigan, USA), LY294002 (PI3K inhibitor), U0126 (ERK/MAPK inhibitor), SB203580 (p38/MAPK inhibitor), SB431542 (TGF- β type I receptor inhibitor), and PD98050 (ERK inhibitor). The primary antibodies used are as follows: Smad2/3, Smad7, slug, pP38 (Santa Cruz Biotechnology, Dallas, TX. USA), snail, vimentin, AKT, pAKT, p38, ERK, pERK, pSmad2, pSmad3, GAPDH (Cell Signaling Technology, Beverly, MA, USA), NOX4 (Novus Biologicals, Littleton, CO, USA) and ZO-1, NOX2 (BD Biosciences, San Diego, CA, USA).
Cell culture treatment
HeLa cells were cultured in DMEM supplemented with 10% FBS and 1% Penicillin/Streptomycin at standard culture conditions (37 °C and 5% CO2). After reaching 70% confluency, the cells were treated with TGF-β (5 ng/mL) for 24 h. For time-course analyses, time points of 0 h, 3 h, 6 h, 12 h, and 24 h after TGF-β treatment were taken into consideration. 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 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 EMT and NOX.
Using a 200 µL pipette tip, a scratch was made in cell monolayers with more than 80% confluence. The cell debris formed during the in vitro wounding process was washed away using 1X phosphate buffer saline (PBS). Furthermore, we pretreated the cells for a period of 1 h with NAC (1 mM), APO (10 µM), DPI (5 µM), and GKT (10 µM) before incubating the cells with 5 ng/mL of TGF-β for 48 h. Microscopic images [inverted microscope, Olympus CKX41 (Olympus Corporation, Tokyo, Japan)] were taken at 0 h and 48 h after TGF-β addition and the wound closure was calculated using ImageJ software.
Overnight serum-starved (DMEM with 1% FBS) HeLa cells were seeded onto Transwell inserts with 8 µm pore size and immersed in a lower chamber containing DMEM supplemented with 2% FBS, with or without TGF-β (5 ng/mL) for 48 h, and with 1 h pretreatment of NAC (1 mM), APO (10 µM), DPI (5 µM), or GKT (10 µM). At the end of the indicated treatment period, non-migrated cells in the top portion of the insert were removed using a cotton swab, while the invaded cells in the bottom portion of the insert were fixed using 4% paraformaldehyde followed by PBS wash and staining using 1% crystal violet solution. The number of cells invaded per field was calculated using ImageJ software.
Proteins were extracted from the treated cells using RIPA buffer and quantified using the Pierce BCA Protein assay kit (Thermo Scientific, MA, USA). Equal amounts of each protein sample were loaded and separated based on their molecular weight using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to a 0.45 µm nitrocellulose blotting membrane for further detection. After blocking the unoccupied sites in the blotting membrane using 10% skim milk [prepared in 1X Tween 20-Tris buffered saline (TTBS)], the membrane was incubated with primary and secondary antibodies with 4 cycles of 10 min washes using 1X PBS in between. The blot was developed using the Western ECL kit (LPS Solution, Daejeon, Republic of Korea) and quantified using ImageJ software.
List of primers used in PCR analysis to detect factors associated with EMT
Forward (5′ → 3′)
Reverse (5′ → 3′)
All data are represented as mean ± standard error of the mean. Statistical significance was analyzed using t-tests and the value was considered to be significant when P < 0.05.
Results and discussion
As principal cellular secondary messengers, reactive oxygen species (ROS) are involved in a myriad of biological functions including the initiation and advancement of epithelial to mesenchymal transition (EMT) by modulating extracellular matrix (ECM) components, cell-contact junctions, and metastasis induction . However, the precise molecular mechanism and key modulators underlying the involvement of ROS in EMT and cancer progression is still poorly understood. In this study, we investigated the impact of ROS on cell migration and EMT upon induction with TGF-β.
Altogether, these results suggest that EMT and its underlying processes, such as migration and invasion, are mediated by NOX enzymes (NOX2 and NOX4). Drug candidates targeting NOX induction and redox-signal transduction cascade kinases inhibit NOX-mediated EMT progression thereby exhibiting promising results against TGF-β mediated cervical cancer advancement.
YMK performed the experiments. KM assisted YMK in result analysis and wrote the manuscript. YMK and MJC conceived and designed the experiments along with correction of the manuscript. All authors read and approved the final manuscript.
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2017R1C1B2011197).
Compliance with ethical standards
The authors declare that they have no competing interests.
- 16.Vignais M, Fafet P. TGFβ-dependent Epithelial-Mesenchymal Transition. In: Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000–2013. https://www.ncbi.nlm.nih.gov/books/NBK6525/
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