TLR4 promotes the expression of HIF-1α by triggering reactive oxygen species in cervical cancer cells in�vitro-implications for therapeutic intervention

Abstract

The present study investigated the mechanism underlying Toll-like receptor 4 (TLR4)-mediated stimulation of hypoxia-inducible factor-1α (HIF-1α) activity and its association with reactive oxygen species (ROS) in cervical cancer cells. SiHa cells were cultured and randomized to control, lipopolysaccharide (LPS), methyl-β-cyclodextrin (MβCD)+LPS, ammonium pyrrolidinedithiocarbamate (PDTC)+LPS, ST2825+LPS and small interfering (si) RNA TLR4+LPS treatment groups. Cell proliferation was quantified using an MTT assay, cell cloning was performed using soft agar colony formation and HIF-1α expression was detected by immunocytochemical staining and western blot analyses. Dichloro-dihydro-fluorescein diacetate and lucigenin luminescence assays were used to detect alterations in ROS and nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase content, respectively. Co-localization of TLR4 and HIF-1α was detected by immunofluorescence staining and observed using fluorescence microscopy. Compared with the control group, cell proliferation was enhanced in the LPS-treated group and was not altered in the PDTC+LPS treatment group. Cell proliferation was reduced in all other treatment groups (P<0.05). Compared with the LPS group, cell proliferation decreased in all other groups. Compared with the PDTC+LPS treatment group, cell proliferation significantly decreased when LPS was co-administered with ST2825, siTLR4 and MβCD (P<0.01). Treatment with MβCD+LPS exhibited an increased inhibitory effect on cell activity and proliferation. Compared with the control group, HIF-1α expression was enhanced following treatment with LPS, although it decreased when LPS was co-administered with ST2825, siTLR4 and MβCD (P<0.05). HIF-1α expression decreased following treatment with ST2825, siTLR4, MβCD and PDTC+LPS, compared with treatment with LPS alone. Compared with the PDTC+LPS group, HIF-1α activity decreased when LPS was co-administered with ST2825, siTLR4 and MβCD. NADPH oxidase and ROS levels increased in cells treated with LPS, compared with the control group, at 24 and 12 h following treatment, respectively, and decreased at 12 h when LPS was co-administered with ST2825, siTLR4 and MβCD. There was no difference between the LPS and PDTC+LPS groups with respect to NADPH and ROS levels. Compared with the PDTC+LPS group, NADPH oxidase activity and ROS content decreased when LPS was co-administered with ST2825, siTLR4 and MβCD. NADPH oxidase activity and ROS content were lowest in the MβCD+LPS treatment group, and immunofluorescent staining demonstrated that TLR4 was localized to the cell surface and HIF-1α was primarily localized to the cytoplasm. TLR4 was co-expressed with HIF-1α in cervical cancer cells. The results of the present study suggested that TLR4 signaling primarily promoted HIF-1α activity via activation of lipid rafts/NADPH oxidase redox signaling and may be associated with the initiation and progression of cervical cancer. This promoting effect was stronger in TLR4/lipid rafts/NADPH oxidase pathway than that in TLR4-NF-κB sig...

Pathogenic role of exosomes and microRNAs in HPV‐mediated inflammation and cervical cancer: A review

“…TLR4 signaling initiates cell activation, inflammation and tumor growth through MyD88‐dependent and NF‐κB‐associated signaling pathways. TLR4 downregulation has been demonstrated to be a potential cause of apoptosis in SiHa CC cells . Furthermore, epidermal growth factor receptor (EGFR) contributes to extensive crosstalk between other signaling pathways, which are involved in the release of growth factors, cytokines and inflammatory mediators.…”

“…TLR4 downregulation has been demonstrated to be a potential cause of apoptosis in SiHa CC cells. 42 Furthermore, epidermal growth factor receptor (EGFR) contributes to extensive crosstalk between other signaling pathways, which are involved in the release of growth factors, cytokines and inflammatory mediators. EGFR overexpression has been found to be related with the induction of inflammation and with poor prognosis in CC patients.…”

“…EGFR overexpression has been found to be related with the induction of inflammation and with poor prognosis in CC patients. 42 Cytokines are small, secreted proteins that are released by cells. Cytokine is a general term for secreted mediators of cell-cell communication.…”

Downregulation of Inflammatory Cytokine Release from IL-1β and LPS-Stimulated PBMC Orchestrated by ST2825, a MyD88 Dimerisation Inhibitor

“…Several studies have reported that ST2825 causes decreased recruitment and activation of specific molecules and transcription factors involved in the activation of TLR4 and the LPS-dependent immune response activation. The main inhibited molecules, due to the activity of ST2825, are IRAK1, IRAK4, TRAF6, p-IKK, p-IkBα, p-NF-κB and HIF-1α [ 48 , 49 ]. ST2825 has also been proposed as a novel drug targeting in diseases like lymphoma, leukaemia, human hepatocellular carcinoma, and traumatic brain injury [ 50 , 51 , 52 ].…”

Clinical and Genetic Landscape of Treatment Naive Cervical Cancer: Alterations in <i>PIK3CA</i> and in Epigenetic Modulators Associated with Sub-Optimal Outcome

“…Four hundred and nineteen patients with histologically proven cervical carcinoma cases were prospectively recruited between 2013 and 2016 in 18 centers from seven European countries [7,8]. Fourty two patients were not eligible for analysis due to: inclusion error [26], death before start of treatment [1], investigator/patient decision [6], second cancer [4], lost to follow up [3], or missing data [2]. At a median follow up time of 22 months [min = 1•1max = 41•2], PFS data is available for 377 patients.…”