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. 2021 May 14;12(5):490.
doi: 10.1038/s41419-021-03771-z.

Cephalomannine inhibits hypoxia-induced cellular function via the suppression of APEX1/HIF-1α interaction in lung cancer

Asmat Ullah  1 , Sze Wei Leong  2 , Jingjing Wang  1 , Qing Wu  1 , Mohsin Ahmad Ghauri  1 , Ammar Sarwar  1 , Qi Su  3 , Yanmin Zhang  4
Affiliations

Cephalomannine inhibits hypoxia-induced cellular function via the suppression of APEX1/HIF-1α interaction in lung cancer

Asmat Ullah et al. Cell Death Dis. .

Abstract

Lung cancer (LC) is one of the leading causes of cancer-related death. As one of the key features of tumor microenvironment, hypoxia conditions are associated with poor prognosis in LC patients. Upregulation of hypoxic-induced factor-1α (HIF-1α) leads to the activation of various factors that contribute to the increased drug resistance, proliferation, and migration of tumor cells. Apurinic/apyrimidinic endonuclease-1 (APEX1) is a multi-functional protein that regulates several transcription factors, including HIF-1α, that contribute to tumor growth, oxidative stress responses, and DNA damage. In this study, we explored the mechanisms underlying cell responses to hypoxia and modulation of APEX1, which regulate HIF-1α and downstream pathways. We found that hypoxia-induced APEX1/HIF-1α pathways regulate several key cellular functions, including reactive oxygen species (ROS) production, carbonic anhydrase 9 (CA9)-mediated intracellular pH, migration, and angiogenesis. Cephalomannine (CPM), a natural compound, exerted inhibitory effects in hypoxic LC cells via the inhibition of APEX1/HIF-1α interaction in vitro and in vivo. CPM can significantly inhibit cell viability, ROS production, intracellular pH, and migration in hypoxic LC cells as well as angiogenesis of HUVECs under hypoxia through the inhibition of APEX1/HIF-1α interaction. Taken together, CPM could be considered as a promising compound for LC treatment.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Cephalomannine (CPM) inhibits cell growth and hypoxia-induced APEX1/HIF-1α pathways in hypoxic LC cells.
A H460 and A549 were treated with and without CPM under normoxia and hypoxia, and cell viability was examined at 24, 48, and 72 h. Data were expressed as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 compared with control cells. The results shown are representative of three independent experiments. B Hypoxia-induced APEX1/HIF-1α pathways in LC cells in vitro. Cell lines were cultured in normoxia and 1% O2 for 0, 12, and 24 h. HIF-1α, APEX1, CA9, CXCR4, and EPO mRNA expression values were assessed by RT-qPCR. Gene expression is normalized to ACTB. Data were expressed as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 compared with control cells. C H460 and H1299 cells were incubated in 1% O2 for 0, 12, and 24 h. Protein levels of HIF-1α and APEX1 were determined by western blotting. β-Actin were used as a control for western blots. n = 3. D H460 cells were cultured under hypoxia. APEX1 (green), HIF-1α (red), DAPI (blue) staining, and 3-channel merged images indicated the nuclear colocalization of HIF-1α. Scale bars, 50 μm. Data presented are representative of three independent experiments.
Fig. 2
Fig. 2. CPM inhibited APEX1/HIF-1α expression in hypoxic LC cells in vitro.
A RT-qPCR analysis of APEX1 and HIF-1α expression in H460 and A549 cells with or without CPM during hypoxia and normoxia. B H460 and A549 cells were treated with CPM and cultured under 1% O2. HIF-1α and APEX1 protein levels were determined by western blotting. β-Actin was used as a control. C H460 cells were treated with CPM, 10 μM APEX1 inhibitor E3330, and 5 μM LW6 in normoxia and hypoxia. Protein expression of HIF-1α was assessed by western blotting. D Relative expression of CA9, CXCR4, and EPO in H460 cells treated with CPM in normoxia and hypoxia. Data were expressed as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 compared as indicated.
Fig. 3
Fig. 3. CPM inhibited APEX1/HIF-1α interaction and reactive oxygen species (ROS) in LC cells.
A Cell extracts from H460 cells treated with CPM under hypoxia were immunoprecipitated with anti-APEX1 antibody or IgG. The IP complex was analyzed for HIF-1α or APEX1 by western blotting. B H460 cells were treated with CPM under hypoxia. Intracellular localization of APEX1 and HIF-1α was examined by immunofluorescence. APEX1 (green), HIF-1α (red), DAPI (blue) staining, and 3-channel merged images indicated the nuclear colocalization. C H460 and A549 cells were treated with E3330 under normoxia and hypoxia. D H460 and A549 LC cells were treated with LW6 under normoxia and hypoxia. E LC cells H460 and A549 treated with CPM under normoxia and hypoxia. Cells were stained with 10 μM DCFH-DA and measured with a flow cytometer.
Fig. 4
Fig. 4. CPM could acidify intracellular pH in LC cells during hypoxia.
A Western blot analysis of CA9 expression at various concentrations treated with CPM. B Intracellular pH determination of H460 cell line treated with APEX1 inhibitor E3330 under normoxia and hypoxia using flow cytometry. C Intracellular pH determination of H460 cells line treated with LW6 under normoxia and hypoxia using flow cytometry. D Intracellular pH determination of H460 cell line-treated CPM under normoxia and hypoxia using flow cytometry, using BCECF-AM dye for staining in H460 cells, images taken by a fluorescence microscope.
Fig. 5
Fig. 5. Inhibitory effect of CPM on migration in LC cells.
A CXCR4, MMP2, and MMP9 expression was analyzed in hypoxic-exposed H460 and A549 cells and treated with CPM at various concentrations. B Hypoxia-induced migration in H460 and A549 cells was examined by transwell assay. H460 and A549 cells were cultured in the upper partition of a transwell chamber exposed to 1% O2, and treated with APEX1 inhibitor E3330, HIF-1α inhibitor LW6 and CPM. Scale bar, 50 μm. C Wound healing assay was performed to test the effect of CPM on cell migration. H460 and A549 cells were incubated with E3330, LW6, and CPM, and exposed to 1% O2 for 48 h. Scale bar, 100 μm. The results shown were representative of three independent experiments.
Fig. 6
Fig. 6. CPM inhibited the tube formation in HUVECs.
A HUVEC cell viability assay by treating with 5 μM BAY-85-3934, 25 nM CPM, 10 μM E3330, and 5 μM LW6. B HUVECs cells were cultured in normoxia and exposed to 1% O2 and also treated with BAY-85-3934 in 1% O2. C One percent O2-exposed HUVECs were treated with CPM, E3330, and LW6. Scale bar, 100 μm. Data were expressed as mean ± SEM (n = 3). **P < 0.01, ***P < 0.001 compared to control groups.
Fig. 7
Fig. 7. CPM inhibited growth and proliferation of H460 LC xenograft tumor model.
A Xenograft tumor volume was measured in the control group and the CPM-treated group during the 10-day experiment. B Xenografts tumor mass was measured of control and CPM-treated group. C Image of tumors from the control group and the CPM-treated group in a xenograft tumor model. D Analyzing the tumor tissue proliferation, both control and CPM-treated tissue were stained with proliferation marker Ki67. Scale bar, 50 μm. E Xenograft animals body weight was measured in the control group and the CPM-treated group during the 10-day experiment. F Western blot analysis of APEX1 and HIF-1α in control group tumor tissue and CPM-treated tumor of xenograft tumor. G Western blotting analysis of protein expression of CA9, CXCR4, MMP2, and MMP9 in control group tumor tissue and CPM-treated tumor of xenograft tumor. H Immunohistochemical staining of CA9, CXCR4, MMP2, and MMP9 in the section of the control group and the CPM-treated group of xenografts nude mice. I Weight of the vital organs heart, liver, spleen, lung, and kidney of animals of the control group and the CPM-treated group. Data were expressed as mean ± SEM (n = 5). *P < 0.05, compared to control groups.
Fig. 8
Fig. 8. CPM inhibited hypoxic-induced HIF-1α/APEX1 interaction in H460 LC xenograft tumor model in vivo.
A Molecular docking model of CPM with APEX1 protein (PDB: 3U8U) and HIF-1α protein (PDB: 4H6J). (I) 3D conformation position of CPM in the surface pocket. (II) Key interactions of CPM in the surface pocket. B Molecular docking model of HIF-1α peptide with APEX1 protein (PDB: 3U8U). (I) 3D conformation position of HIF-1α peptide in the surface pocket of APEX1. (II) Key interactions of HIF-1α peptide in the surface pocket of APEX1. C Overlapping of docking conformations of CPM and HIF-1α peptide in APEX1 protein. D Immunofluorescence staining of H460 tumor sections. Tumor section were stained with HIF-1α (green), APEX1 (red), and DAPI (blue).
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