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

The present study investigated the mechanism underlying Toll-like receptor 4 (TLR4)-mediated stimulation of hypoxia-inducible factor-1 (HIF-1) activity and its association with reactive oxygen species (ROS) in cervical cancer cells. SiHa cells were cultured and randomized to control, lipopolysaccharide (LPS), methyl-beta-cyclodextrin (MCD)+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-1 alpha 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-1 alpha 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 M beta CD (P<0.01). Treatment with M beta CD+LPS exhibited an increased inhibitory effect on cell activity and proliferation. Compared with the control group, HIF-1 alpha expression was enhanced following treatment with LPS, although it decreased when LPS was co-administered with ST2825, siTLR4 and M beta CD (P<0.05). HIF-1 alpha expression decreased following treatment with ST2825, siTLR4, M beta CD and PDTC+LPS, compared with treatment with LPS alone. Compared with the PDTC+LPS group, HIF-1 alpha activity decreased when LPS was co-administered with ST2825, siTLR4 and M beta CD. 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 M beta CD. 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 M beta CD. NADPH oxidase activity and ROS content were lowest in the M beta CD+LPS treatment group, and immunofluorescent staining demonstrated that TLR4 was localized to the cell surface and HIF-1 alpha was primarily localized to the cytoplasm. TLR4 was co-expressed with HIF-1 alpha in cervical cancer cells. The results of the present study suggested that TLR4 signaling primarily promoted HIF-1 alpha 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-kappa B signaling pathway. Therefore, the TLR4/lipid raft-associated redox signal may be a target for therapeutic intervention to prevent the growth of cervical cancer.