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. 2018 Feb;17(2):2229-2238.
doi: 10.3892/mmr.2017.8108. Epub 2017 Nov 20.

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

Xiao Yang  1 , Gan Tao Chen  2 , Yan Qing Wang  1 , Shu Xian  1 , Li Zhang  1 , Shao Ming Zhu  3 , Feng Pan  4 , Yan Xiang Cheng  1
Affiliations

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

Xiao Yang et al. Mol Med Rep. 2018 Feb.

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 signaling pathway. Therefore, the TLR4/lipid raft-associated redox signal may be a target for therapeutic intervention to prevent the growth of cervical cancer.

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Figures

Figure 1.
Figure 1.
An MTT assay was used to measure cell growth in the groups following 1, 2, 3 and 4 days of treatment. Data are presented as the mean ± standard deviation (n=3). **P<0.01 vs. the control group; #P<0.05, ##P<0.01, vs. the LPS group; &&P<0.01 vs. the PDTC+LPS group. LPS, lipopolysaccharide; si, small interfering RNA; TLR4, Toll-like receptor 4; MβCD, methyl-β-cyclodextrin; PDTC, ammonium pyrrolidinedithiocarbamate.
Figure 2.
Figure 2.
Soft agar colony forming experiment. (A) Images of soft agar plate colony formation for treatment groups following 2 weeks of culture. (B) Quantification of colony numbers. Data are presented as the mean ± standard deviation (n=3). *P<0.05 and **P<0.01 vs. the control group; #P<0.05, ##P<0.01 vs. the LPS group; &P<0.05 and &&P<0.01 vs. the PDTC+LPS group. LPS, lipopolysaccharide; si, small interfering RNA; TLR4, Toll-like receptor 4; MβCD, methyl-β-cyclodextrin; PDTC, ammonium pyrrolidinedithiocarbamate.
Figure 3.
Figure 3.
HIF-1α expression levels. (A) Immunocytochemistry images (magnification, ×100). Positive cells were stained brown-yellow. (B) Western blot analysis of HIF-1α expression levels. (C) Statistical analysis of western blotting. The gel was cut to improve the clarity of the figure, as shown by a dotted line. Data are presented as the mean ± standard deviation (n=3). *P<0.05 and **P<0.01 vs. the control group; #P<0.05, ##P<0.01 vs. the LPS group; &P<0.05 and &&P<0.01 vs. the PDTC+LPS group. LPS, lipopolysaccharide; si, small interfering RNA; TLR4, Toll-like receptor 4; MβCD, methyl-β-cyclodextrin; PDTC, ammonium pyrrolidinedithiocarbamate.
Figure 4.
Figure 4.
Detection of NADPH oxidase activity in treatment groups at 0, 12, 24, 36 and 48 h. *P<0.05 and **P<0.01 vs. the control group at 0, 12, 24, 36 and 48 h. #P<0.05, ##P<0.01 vs. the LPS group at 0, 12, 24, 36 and 48 h. &P<0.05 and &&P<0.01 vs. the PDTC+LPS group at 0, 12, 24, 36 and 48 h. LPS, lipopolysaccharide; si, small interfering RNA; TLR4, Toll-like receptor 4; MβCD, methyl-β-cyclodextrin; PDTC, ammonium pyrrolidinedithiocarbamate; nicotinamide-adenine dinucleotide phosphate; NADPH, nicotinamide-adenine dinucleotide phosphate; RLU, relative light units.
Figure 5.
Figure 5.
ROS fluorescence intensity of each group detected at 0, 12, 24, 36 and 48 h. *P<0.05 and **P<0.01 vs. the control group at 0, 12, 24, 36 and 48 h. ##P<0.01, vs. the LPS group at 0, 12, 24, 36 and 48 h. &&P<0.01 vs. the PDTC+LPS group at 0, 12, 24, 36 and 48 h. LPS, lipopolysaccharide; si, small interfering RNA; TLR4, Toll-like receptor 4; MβCD, methyl-β-cyclodextrin; PDTC, ammonium pyrrolidinedithiocarbamate; ROS, reactive oxygen species.
Figure 6.
Figure 6.
The expression and co-localization of TLR4 and HIF-1α detected by immunofluorescence staining. Green, HIF-1α expression; red, TLR4 expression; blue, nuclei. LPS, lipopolysaccharide; si, small interfering RNA; TLR4, Toll-like receptor 4; MβCD, methyl-β-cyclodextrin; PDTC, ammonium pyrrolidinedithiocarbamate.
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