Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Nov 8;19(1):1075.
doi: 10.1186/s12885-019-6312-y.

Aurora-a confers radioresistance in human hepatocellular carcinoma by activating NF-κB signaling pathway

Ze-Tian Shen  1 , Ying Chen  2 , Gui-Chun Huang  2 , Xi-Xu Zhu  1 , Rui Wang  3 , Long-Bang Chen  4
Affiliations

Aurora-a confers radioresistance in human hepatocellular carcinoma by activating NF-κB signaling pathway

Ze-Tian Shen et al. BMC Cancer. .

Abstract

Background: Radiotherapy failure is a significant clinical challenge due to the development of resistance in the course of treatment. Therefore, it is necessary to further study the radiation resistance mechanism of HCC. In our early study, we have showed that the expression of Aurora-A mRNA was upregulated in HCC tissue samples or cells, and Aurora-A promoted the malignant phenotype of HCC cells. However, the effect of Aurora-A on the development of HCC radioresistance is not well known.

Methods: In this study, colony formation assay, MTT assays, flow cytometry assays, RT-PCR assays, Western blot, and tumor xenografts experiments were used to identify Aurora-A promotes the radioresistance of HCC cells by decreasing IR-induced apoptosis in vitro and in vivo. Dual-luciferase reporter assay, MTT assays, flow cytometry assays, and Western blot assay were performed to show the interactions of Aurora-A and NF-κB.

Results: We established radioresistance HCC cell lines (HepG2-R) and found that Aurora-A was significantly upregulated in those radioresistant HCC cells in comparison with their parental HCC cells. Knockdown of Aurora-A increased radiosensitivity of radioresistant HCC cells both in vivo and in vitro by enhancing irradiation-induced apoptosis, while upregulation of Aurora-A decreased radiosensitivity by reducing irradiation-induced apoptosis of parental cells. In addition, we have showed that Aurora-A could promote the expression of nuclear IkappaB-alpha (IκBα) protein while enhancing the activity of NF-kappaB (κB), thereby promoted expression of NF-κB pathway downstream effectors, including proteins (Mcl-1, Bcl-2, PARP, and caspase-3), all of which are associated with apoptosis.

Conclusions: Aurora-A reduces radiotherapy-induced apoptosis by activating NF-κB signaling, thereby contributing to HCC radioresistance. Our results provided the first evidence that Aurora-A was essential for radioresistance in HCC and targeting this molecular would be a potential strategy for radiosensitization in HCC.

Keywords: Apoptosis; Aurora-a; Hepatocellular carcinoma; NF-kappaB; Radioresistance.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no competing interest.

Figures

Fig. 1
Fig. 1
Establishment of radioresistant HCC cells. a A representative image of colony formation in parental and their radioresistant HCC cells treated with various dose of IR (0.0, 4.0 and 6.0 Gy) after 14 days. b The results of colony formation. c Survival fractions of parental and their radioresistant HCC cells were obtained from the results of the colony-forming assays. d Flow cytometric detection of apoptosis in parental and their radioresistant HCC cells treated with various dose of IR (0.0, 4.0 and 6.0 Gy). Data represent the mean ± S.E. of three individual experiments with triplicates. *p < 0.05 and**p < 0.01
Fig. 2
Fig. 2
Effect of Aurora-A knockdown on in vitro radiosensitivity of radioresistant HCC cells. a RT-PCR and Western blot was used to detect the Aurora-A mRNA and protein expression in radioresistant and their parental HCC cells. The internal control was GAPDH. b RT-PCR and Western blot was used to detect the Aurora-A mRNA and protein expression in the stably transfected HCC cells (HepG2-R/shAuro or HepG2-R/shcontrol). The internal control was GAPDH. c A representative image of colony formation in the stably tansfected HCC cells treated with various dose of IR (0.0 and 4.0 Gy) after 14 days. d Survival fractions of the stably transfected HCC cells were obtained from the results of the MTT assays. e Flow cytometric detection of apoptosis in the stably transfected HCC cells treated with various dose of IR (0.0 and 4.0Gy). Data represent the mean ± S.E. of three individual experiments with triplicates. *p < 0.05 and**p < 0.01
Fig. 3
Fig. 3
Effect of Aurora-A upregualtion on in vitro radiosensitivity of parental HCC cells. a RT-PCR and Western blot detection of Aurora-A mRNA and protein expression in the stably transfected HCC cells (HepG2/Auro or HepG2/control). The internal control was GAPDH. b A representative image of colony formation in the stably transfected HCC cells treated with various dose of IR (0.0 and 4.0 Gy) after 14 days. c Survival fractions of the stably transfected HCC cells were obtained from the results of the MTT assays. d Flow cytometric detection of apoptosis in the stably transfected HCC cells treated with various dose of IR (0.0 and 4.0Gy). Data represent the mean ± S.E. of three individual experiments with triplicates. *p < 0.05 and**p < 0.01
Fig. 4
Fig. 4
Effect of Aurora-A knockdown on in vivo radiosensitivity of radioresistant HCC cells. a The growth of subcutaneous tumor derived from HepG2-R/shAuro and HepG2-R/shcontrol cells in BALB/c athymic nude mice. Mice were treated with 8.0 Gy irradiation at seventh day post tumor cell injection. Five mice were inoculated. b Representative features of tumors 42d after inoculation using HepG2-R/shAuro and HepG2-R/shcontrol cells treated with IR. c Western blotting was used to detect the Aurora-A protein expression in tumors developed from HepG2-R/shAuro and HepG2-R/shcontrol cells treated with IR, respectively. The internal control was GAPDH. d Immunostaining of PCNA protein expression in tumors developed from HepG2-R/shAuro and HepG2-R/shcontrol cells treated with IR. Lower: immunostaining; Upper: H&E staining; Bars, 100 μm. e TUNEL assay was used to detect the apoptosis in tumors developed from HepG2-R/shAuro and HepG2-R/shcontrol cells treated with IR, respectively. Data represent the mean ± S.E. of three individual experiments with triplicates. *p < 0.05 and**p < 0.01
Fig. 5
Fig. 5
Effect of Aurora-A upregulation on in vivo radiosensitivity of parental HCC cells. a The growth of subcutaneous tumor derived from HepG2/Auro and HepG2/control cells in BALB/c athymic nude mice. Mice were treated with 8.0Gy irradiation at seventh day post tumor cell injection. Five mice were inoculated. b Representative features of tumors 42d after inoculation using HepG2/Auro and HepG2/control cells treated with IR. c Western blotting was used to detect the Aurora-A protein expression in tumors developed from HepG2/Auro and HepG2/control cells treated with IR, respectively. The internal control was GAPDH. d Immunostaining of PCNA protein expression in tumors developed from HepG2/Auro and HepG2/control cells treated with IR. Lower: immunostaining; Upper: H&E staining; Bars, 100 μm. e TUNEL assay was used to detect the apoptosis in tumors developed from HepG2/Auro and HepG2/control cells treated with IR, respectively. Data represent the mean ± S.E. of three individual experiments with triplicates. *p < 0.05 and**p < 0.01
Fig. 6
Fig. 6
Aurora-a promotes the activation of NF-κB signaling in HCC cells. a Western blotting was used to detect the nuclear or cytoplasmic IκBα protein expression in parental and their radioresistant HCC cells. The internal control was GAPDH or Topo I, respectively. b Western blotting detection of p65 protein expression in parental and their radioresistant HCC cells. The internal control was GAPDH. c A luciferase reporter system was used to measure the activity of NF-κB in parental and their radioresistant HCC cells. d Western blotting was used to detect the nuclear or cytoplasmic IκBα protein and p65 expression in the Aurora-A-knockdown HCC cells. The internal control was GAPDH or Topo I, respectively. e Western blotting was used to detect the nuclear or cytoplasmic IκBα protein and p65 expression in the Aurora-A-overexpressing HCC cells. The internal control was GAPDH or Topo I, respectively. f A luciferase reporter system was used to measure the activity of NF-κB in the stably transfected HCC cells. g and h Western blotting was used to detect the apoptosis-related proteins (Bcl-2, Mcl-1, cleaved PARP and caspase-3) in the Aurora-A-knockdown and Aurora-A-overexpressing HCC cells. The internal control was GAPDH. Data represent the mean ± S.E. of three individual experiments with triplicates. N.S, p > 0.05, *p < 0.05 and**p < 0.01
Fig. 7
Fig. 7
NF-κB signaling was involved in Aurora-A-promoting radioresistance in HCC cells. a MTT assays were employed to evaluate survival fractions of the stably transfected HCC cells plus LPS and IR (4.0Gy). b A representative image of colony formation in the stably transfected HCC cells plus LPS and IR (4.0Gy) after 14 days. c Flow cytometric detection of apoptosis in the stably transfected HCC cells plus LPS and IR (4.0Gy). d Western blot detection of the expression of those proteins (p65, Bcl-2, Mcl-1, cleaved PARP and caspase-3) in the stably transfected HCC cells plus LPS. GAPDH was used as an internal control. Data represent the mean ± S.E. of three individual experiments with triplicates. *p < 0.05 and**p < 0.01
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin. 2018;68(1):7–30. doi: 10.3322/caac.21442. - DOI - PubMed
    1. Pascual S, Herrera I, Irurzun J. New advances in hepatocellular carcinoma. World J Hepatol. 2016;8(9):421–438. doi: 10.4254/wjh.v8.i9.421. - DOI - PMC - PubMed
    1. Kulik LM, Carr BI, Mulcahy MF, Lewandowski RJ, Atassi B, Ryu RK, Sato KT, Benson A, 3rd, Nemcek AA, Jr, Gates VL, et al. Safety and efficacy of 90Y radiotherapy for hepatocellular carcinoma with and without portal vein thrombosis. Hepatology. 2008;47(1):71–81. doi: 10.1002/hep.21980. - DOI - PubMed
    1. Mornex F, Girard N, Beziat C, Kubas A, Khodri M, Trepo C, Merle P. Feasibility and efficacy of high-dose three-dimensional-conformal radiotherapy in cirrhotic patients with small-size hepatocellular carcinoma non-eligible for curative therapies--mature results of the French phase II RTF-1 trial. Int J Radiat Oncol Biol Phys. 2006;66(4):1152–1158. doi: 10.1016/j.ijrobp.2006.06.015. - DOI - PubMed
    1. Ducat D, Zheng Y. Aurora kinases in spindle assembly and chromosome segregation. Exp Cell Res. 2004;301(1):60–67. doi: 10.1016/j.yexcr.2004.08.016. - DOI - PubMed

MeSH terms