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 Jan 27:2019:4012590.
doi: 10.1155/2019/4012590. eCollection 2019.

Inhibition of LOXL2 Enhances the Radiosensitivity of Castration-Resistant Prostate Cancer Cells Associated with the Reversal of the EMT Process

Peng Xie  1 , Hongliang Yu  1 , Feijiang Wang  1 , Feng Yan  2 , Xia He  1
Affiliations

Inhibition of LOXL2 Enhances the Radiosensitivity of Castration-Resistant Prostate Cancer Cells Associated with the Reversal of the EMT Process

Peng Xie et al. Biomed Res Int. .

Abstract

Introduction: Radiotherapy is the mainstay in the treatment of prostate cancer. However, significant radioresistance of castration-resistant prostate cancer (CRPC) cells constitutes a main obstacle in the treatment of this disease. By using bioinformatic data mining methods, LOXL2 was found to be upregulated in both androgen-independent prostate cancer cell lines and radioresistant tumor samples collected from patients with prostate cancer. We speculate that LOXL2 may play an important role in the radioresistance of CRPC cells.

Methods: The effect of LOXL2 knockdown on the radiosensitivity of androgen-independent prostate cancer cells lines was measured by the clonogenic assay and xenograft tumor experiments under in vitro and in vivo conditions, respectively. In studies on the mechanism, we focused on the EMT phenotype changes and cell apoptosis changes induced by LOXL2 knockdown in DU145 cells. The protein levels of three EMT biomarkers, namely, E-cadherin, vimentin, and N-cadherin, were measured by western blotting and immunohistochemical staining. Cell apoptosis after irradiation was measured by flow cytometry and caspase-3 activity assay. Salvage experiment was also conducted to confirm the possible role of EMT in the radiosensitization effect of LOXL2 knockdown in CRPC cells.

Results: LOXL2 knockdown in CRPC cells enhanced cellular radiosensitivity under both in vitro and in vivo conditions. A significant reversal of EMT was observed in LOXL2-silenced DU145 cells. Cell apoptosis after irradiation was significantly enhanced by LOXL2 knockdown in DU145 cells. Results from the salvage experiment confirmed the key role of EMT process reversal in the radiosensitization effect of LOXL2 knockdown in DU145 cells.

Conclusions: LOXL2 plays an important role in the development of cellular radioresistance in CRPC cells. Targeting LOXL2 may be a rational avenue to overcome radioresistance in CRPC cells. A LOXL2-targeting strategy for CRPC treatment warrants detailed investigation in the future.

PubMed Disclaimer

Figures

Figure 1
Figure 1
LOXL2 was upregulated in CRPC cell lines and radioresistant prostate cancer samples. (a) Diagrammatic representation of heatmap of DEGs in the TCGA dataset. (b) LOXL2 expression was significantly higher in samples of the radioresistant group (RR) than in the radiosensitive group (RS) from the TCGA dataset. (c) Diagrammatic representation of heatmap of DEGs in prostate cell lines with different androgen dependency status from the GEO dataset. (d) A captured histogram picture from the GEO dataset, which shows that the expression of LOXL2 was upregulated in androgen-independent prostate cell lines. (e) LOXL2 expression was higher in CRPC cells of DU145 and PC3 cells than in androgen-dependent LNCaP and 22Rv1 cells at the mRNA level measured by real-time PCR. (f) LOXL2 expression was higher in DU145 and PC3 cells than in LNCaP and 22Rv1 cells at the protein level. ∗ denotes p <0.05 by Student's t-test.
Figure 2
Figure 2
Knockdown of LOXL2 radiosensitizes DU145 and PC3 cells. (a-b) LOXL2 was significantly silenced in DU145 and PC3 cells by si-LOXL2 transfection at both mRNA and protein levels. (c) The radiosensitization effect of LOXL2 knockdown in DU145 and PC3 cells was determined by the clonogenic assay after 4 Gy of irradiation. (d-e) Dose-survival curve derived from the results of clonogenic assay for DU145 cells; the radiobiological parameters D0, Dq, and N are also listed. ∗ denotes p <0.05 by Student's t-test.
Figure 3
Figure 3
The radiosensitizing effect of LOXL2 knockdown in DU145 cells was associated with the reversal of the EMT phenotype and an increase in cell apoptosis. (a) The typical morphology of DU145 and LOXL2-silenced DU145 cells. (b) Protein expression of three classical EMT biomarkers, namely, E-cadherin, vimentin, and N-cadherin, was determined by western blotting. A significant reversal of the EMT process was observed in LOXL2-knockdown DU145 cells. (c-d) LOXL2 knockdown significantly enhanced cell apoptosis in DU145 cells after a 4 Gy dose of irradiation as measured by Annexin V-FITC/PI double staining. (e) LOXL2 knockdown significantly enhanced cell apoptosis in DU145 cells after a 4 Gy dose of irradiation as measured by the caspase-3 activity assay. ∗ denotes p <0.05 by Student's t-test.
Figure 4
Figure 4
Xenograft-based evaluation of the radiosensitization effect of LOXL2 knockdown in CRPC DU145 cells in vivo. (a) The time course of growth of DU145-scramble and DU145-si-LOXL2 xenograft tumors with or without a local 6 Gy dose of IR treatment. (b) Representative photo of residual tumor of DU145-scramble and DU145-si-LOXL2 cells after a 6 Gy dose of irradiation. (c) IHC staining showed an elevated E-cadherin expression in LOXL2-knockdown xenograft tumor, which represents the reversal of the EMT process in LOXL2-knockdown cells in vivo. ∗ denotes p <0.05 by Student's t-test.
Figure 5
Figure 5
Salvage experiment confirmed that EMT process reversal dominated the mechanism for restoring radiosensitivity in DU145 cells after LOXL2 knockdown. (a) EMT phenotype was coercively restored by simultaneous E-cadherin knockdown in LOXL2-silenced DU145 cells, as represented by the restoration of the expression of vimentin and N-cadherin. (b, c) Coercively restoring the EMT process in LOXL2-silenced DU145 cells restored cellular radioresistance as determined by the clonogenic assay. (d-e) Coercively inducing the EMT process restored cellular radioresistance as determined by cell apoptosis study by Annexin V-FITC/PI double staining. ∗ denotes p <0.05 by Student's t-test.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Torre L. A., Bray F., Siegel R. L., Ferlay J., Lortet-Tieulent J. Global cancer statistics, 2012. CA: A Cancer Journal for Clinicians. 2015;65(2):87–108. doi: 10.3322/caac.21262. - DOI - PubMed
    1. Hamdy F. C., Donovan J. L., Lane J. A., et al. 10-Year outcomes after monitoring, surgery, or radiotherapy for localized prostate cancer. The New England Journal of Medicine. 2016;375(15):1415–1424. doi: 10.1056/nejmoa1606220. - DOI - PubMed
    1. Nakfoor B., Zietman A., Gerweck L., Shipley W. An assessment of the in vitro radiosensitivity of a human prostate cancer cell line: The influence of the hormonal millieu. International Journal of Radiation Oncology Biology Physics. 1994;30:p. 321. doi: 10.1016/0360-3016(94)90910-5. - DOI
    1. Wade C. A., Kyprianou N. Profiling prostate cancer therapeutic resistance. International Journal of Molecular Sciences. 2018;19(3):p. 904. - PMC - PubMed
    1. Kim Y.-M., Kim E.-C., Kim Y. The human lysyl oxidase-like 2 protein functions as an amine oxidase toward collagen and elastin. Molecular Biology Reports. 2011;38(1):145–149. doi: 10.1007/s11033-010-0088-0. - DOI - PubMed

MeSH terms

Substances