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Introduction
Cervical cancer is a key factor associated with morbidity and mortality in women worldwide (1). Infection with human papilloma viruses (HPV) triggers carcinogenesis. Most of the precancerous lesions do not progress to invasive carcinoma, suggesting that HPV is not the only factor contributing to the development of cervical cancer (2,3). However, persistent HPV infection alters the pro-inflammatory cytokine profile, resulting in chronic inflammation and recurrence of cervical cancer (4). Cancer stem cells (CSCs) play a vital role in cancer initiation and metastasis (5). Metastasis results in treatment failure and death (6). Epithelial-mesenchymal transition (EMT) has been implicated as the key factor in CSCs transformation (7,8). EMT has been shown to induce reversion to a CSC-like phenotype, linking CSCs and EMT (9,10).
NF-κB is a classic transcription factor activated by inflammatory stimuli, such as LPS (11), TNF-α (12) and IL-10 (13). Activated NF-κB induces extensive gene expression in immune response (TNF-α), angiogenesis (VEGF), invasion (MMP-9) and EMT (Twist) (14–17). Furthermore, NF-κB, a pleiotropic transcription factor, has been implicated in EMT and metastasis (14–17). In mammary epithelial cells, EMT is upregulated via overexpression of NF-κBp65 (17).
The transcriptional factor TWIST mediates EMT and cancer metastasis (18,19). In uterine cancers, Twist overexpression promotes invasion and metastasis (20–23). However, the role of NF-κB/Twist axis in cervical cancer has not been investigated. In this study, we focused on the role of NF-κB/Twist axis in vitro, by co-treatment of human cervical cancer cell line HeLa with TNF-α and TGF-β.
Materials and methods
Reagents
DMEM was obtained from Gibco, FBS from PAA, trypsin and penicillin-streptomycin from Invitrogen, and TGF-β and TNF-α from Sino Biological (Beijing, China). Anti-E-cadherin and anti-N-cadherin antibodies were supplied by Cell Signaling Technology (Danvers, MA, USA). The following antibodies were purchased from Abcam (Burlingame, CA, USA): anti-Bmi1, anti-Sox2, anti-Oct4, anti-CD133, anti-CD44, anti-ALDH1, anti-NF-κBp65 and anti-Twist1.
Cell culture and EMT morphology
HeLa cells were supplied by the Cell Bank of Chinese Academy of Sciences (Shanghai, China), and cultured as described by López et al (5). Cells were incubated with TNF-α (10.0 ng/ml) for 24 h, TGF-β (5.0 ng/ml) for 6 days or TNF-α (10.0 ng/ml) for 24 h along with TGF-β (5.0 ng/ml) for 6 days. EMT morphology were visualized under a light microscope (Olympus, Japan). The...