Skip to main content
SpringerLink
Log in

CPLX2 Regulates Ferroptosis and Apoptosis Through NRF2 Pathway in Human Hepatocellular Carcinoma Cells

  • Original Article
  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Understanding the principle of regulated cell death (RCD) such as ferroptosis and apoptosis provides opportunities to overcome sorafenib resistance of HCC. Complexin II (CPLX2) is involved in calcium-dependent fusion of vesicles and plasma membrane, and recent studies showed CPLX2 is involved in cancer progression. However, the expression and function of CPLX2 are unclear in hepatocellular carcinoma (HCC). qPCR and western blotting assays were used to detect the levels of CPLX2. MTT and colony formation assays were used to detect cell viability. The contents of iron, ROS, MDA, and GSH were used to evaluate the function of CPLX2 on ferroptosis, while the flow cytometry and TUNEL assays were used to evaluate the role of CPLX2 on apoptosis. Our analysis showed CPLX2 is significantly upregulated in HCC, which predicts poor overall survival (OS), progression-free survival (PFS), relapse-free survival (RFS), and disease-specific survival (DSS) for patients with HCC. Further function enrichment analysis of genes related to CPLX2 showed CPLX2 is involved in the NRF2 pathway. Downregulation of CPLX2 can inhibit NRF2 expression and the transcription of its downstream genes, which confirms that CPLX2 is involved in NRF2 pathway. Cell viability assay showed that ferroptosis and apoptosis inhibitors can reverse the inhibition effect of CPLX2-knockdown on cell survival, respectively. And downregulation of CPLX2 significantly promotes the contents of iron, ROS, and MDA, while inhibiting the GSH level of HCC cell lysate, suggesting CPLX2 involved in ferroptosis. Moreover, downregulation of CPLX2 promotes the apoptosis of HCC cells by flow cytometry and TUNEL assay. And upregulation of NRF2 can partly reverse the inhibitory effect of CPLX2-downregulation on ferroptosis and apoptosis. Finally, we found downregulation of CPLX2 aggravates cell death induced by sorafenib. CPXL2 regulates ferroptosis and apoptosis through NRF2 pathway, and CPLX2 knockdown promotes cell death induced by sorafenib. CPLX2 might be an effective target for therapy patients with HCC.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Data Availability

The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.

References

  1. Liang, C., Zhang, X., Yang, M., & Dong, X. (2019). Recent progress in ferroptosis inducers for cancer therapy. Advanced Materials, 31(51), e1904197. https://doi.org/10.1002/adma.201904197

    Article  CAS  Google Scholar 

  2. Tan, M. L., Ooi, J. P., Ismail, N., Moad, A. I., & Muhammad, T. S. (2009). Programmed cell death pathways and current antitumor targets. Pharmaceutical Research, 26(7), 1547–1560. https://doi.org/10.1007/s11095-009-9895-1

    Article  CAS  Google Scholar 

  3. Bialik, S., Zalckvar, E., Ber, Y., Rubinstein, A. D., & Kimchi, A. (2010). Systems biology analysis of programmed cell death. Trends in Biochemical Sciences, 35(10), 556–564. https://doi.org/10.1016/j.tibs.2010.04.008

    Article  CAS  Google Scholar 

  4. Ouyang, L., Shi, Z., Zhao, S., Wang, F. T., Zhou, T. T., Liu, B., et al. (2012). Programmed cell death pathways in cancer: A review of apoptosis, autophagy and programmed necrosis. Cell Proliferation, 45(6), 487–498. https://doi.org/10.1111/j.1365-2184.2012.00845.x

    Article  CAS  Google Scholar 

  5. Angeli, J. P. F., Shah, R., Pratt, D. A., & Conrad, M. (2017). Ferroptosis inhibition: Mechanisms and opportunities. Trends in Pharmacological Sciences, 38(5), 489–498. https://doi.org/10.1016/j.tips.2017.02.005

    Article  CAS  Google Scholar 

  6. Vanden Berghe, T., Linkermann, A., Jouan-Lanhouet, S., Walczak, H., & Vandenabeele, P. (2014). Regulated necrosis: The expanding network of non-apoptotic cell death pathways. Nature Reviews Molecular Cell Biology, 15(2), 135–147. https://doi.org/10.1038/nrm3737

    Article  CAS  Google Scholar 

  7. Dixon, S. J., Lemberg, K. M., Lamprecht, M. R., Skouta, R., Zaitsev, E. M., Gleason, C. E., et al. (2012). Ferroptosis: An iron-dependent form of nonapoptotic cell death. Cell, 149(5), 1060–1072. https://doi.org/10.1016/j.cell.2012.03.042

    Article  CAS  Google Scholar 

  8. Li, J., Cao, F., Yin, H. L., Huang, Z. J., Lin, Z. T., Mao, N., et al. (2020). Ferroptosis: Past, present and future. Cell Death & Disease, 11(2), 88. https://doi.org/10.1038/s41419-020-2298-2

    Article  Google Scholar 

  9. Koppula, P., Zhuang, L., & Gan, B. (2020). Cystine transporter SLC7A11/xCT in cancer: Ferroptosis, nutrient dependency, and cancer therapy. Protein & Cell. https://doi.org/10.1007/s13238-020-00789-5

    Article  Google Scholar 

  10. Kang, R., Kroemer, G., & Tang, D. (2019). The tumor suppressor protein p53 and the ferroptosis network. Free Radical Biology & Medicine, 133, 162–168. https://doi.org/10.1016/j.freeradbiomed.2018.05.074

    Article  CAS  Google Scholar 

  11. Xie, Y., Hou, W., Song, X., Yu, Y., Huang, J., Sun, X., et al. (2016). Ferroptosis: Process and function. Cell Death and Differentiation, 23(3), 369–379. https://doi.org/10.1038/cdd.2015.158

    Article  CAS  Google Scholar 

  12. Savaskan, N. E., Ufer, C., Kuhn, H., & Borchert, A. (2007). Molecular biology of glutathione peroxidase 4: From genomic structure to developmental expression and neural function. Biological Chemistry, 388(10), 1007–1017. https://doi.org/10.1515/BC.2007.126

    Article  CAS  Google Scholar 

  13. Xu, T., Ding, W., Ji, X., Ao, X., Liu, Y., Yu, W., et al. (2019). Molecular mechanisms of ferroptosis and its role in cancer therapy. Journal of Cellular and Molecular Medicine, 23(8), 4900–4912. https://doi.org/10.1111/jcmm.14511

    Article  Google Scholar 

  14. Dodson, M., Castro-Portuguez, R., & Zhang, D. D. (2019). NRF2 plays a critical role in mitigating lipid peroxidation and ferroptosis. Redox Biology, 23, 101107. https://doi.org/10.1016/j.redox.2019.101107

    Article  CAS  Google Scholar 

  15. Rojo de la Vega, M., Chapman, E., & Zhang, D. D. (2018). NRF2 and the hallmarks of cancer. Cancer Cell., 34(1), 21–43. https://doi.org/10.1016/j.ccell.2018.03.022

    Article  CAS  Google Scholar 

  16. Niture, S. K., & Jaiswal, A. K. (2012). Nrf2 protein up-regulates antiapoptotic protein Bcl-2 and prevents cellular apoptosis. Journal of Biological Chemistry, 287(13), 9873–9886. https://doi.org/10.1074/jbc.M111.312694

    Article  CAS  Google Scholar 

  17. Niture, S. K., & Jaiswal, A. K. (2013). Nrf2-induced antiapoptotic Bcl-xL protein enhances cell survival and drug resistance. Free Radical Biology & Medicine, 57, 119–131. https://doi.org/10.1016/j.freeradbiomed.2012.12.014

    Article  CAS  Google Scholar 

  18. Tsuru, E., Oryu, K., Sawada, K., Nishihara, M., & Tsuda, M. (2019). Complexin 2 regulates secretion of immunoglobulin in antibody-secreting cells. Immun Inflamm Dis., 7(4), 318–325. https://doi.org/10.1002/iid3.276

    Article  CAS  Google Scholar 

  19. Komatsu, H., Kakehashi, A., Nishiyama, N., Izumi, N., Mizuguchi, S., Yamano, S., et al. (2013). Complexin-2 (CPLX2) as a potential prognostic biomarker in human lung high grade neuroendocrine tumors. Cancer Biomarkers, 13(3), 171–180. https://doi.org/10.3233/CBM-130336

    Article  CAS  Google Scholar 

  20. Makuuchi, R., Terashima, M., Kusuhara, M., Nakajima, T., Serizawa, M., Hatakeyama, K., et al. (2017). Comprehensive analysis of gene mutation and expression profiles in neuroendocrine carcinomas of the stomach. Biomedical Research, 38(1), 19–27. https://doi.org/10.2220/biomedres.38.19

    Article  CAS  Google Scholar 

  21. Bikkavilli, R. K., Zerayesus, S. A., Van Scoyk, M., Wilson, L., Wu, P. Y., Baskaran, A., et al. (2017). K-homology splicing regulatory protein (KSRP) promotes post-transcriptional destabilization of Spry4 transcripts in non-small cell lung cancer. Journal of Biological Chemistry, 292(18), 7423–7434. https://doi.org/10.1074/jbc.M116.757906

    Article  CAS  Google Scholar 

  22. Yang, H., Cho, M. E., Li, T. W., Peng, H., Ko, K. S., Mato, J. M., et al. (2013). MicroRNAs regulate methionine adenosyltransferase 1A expression in hepatocellular carcinoma. The Journal of Clinical Investigation, 123(1), 285–298. https://doi.org/10.1172/JCI63861

    Article  CAS  Google Scholar 

  23. Latunde-Dada, G. O. (2017). Ferroptosis: Role of lipid peroxidation, iron and ferritinophagy. Biochimica et Biophysica Acta - General Subjects, 1861(8), 1893–1900. https://doi.org/10.1016/j.bbagen.2017.05.019

    Article  CAS  Google Scholar 

  24. Song, Y., Miao, Y., & Song, C. P. (2014). Behind the scenes: The roles of reactive oxygen species in guard cells. New Phytologist, 201(4), 1121–1140. https://doi.org/10.1111/nph.12565

    Article  CAS  Google Scholar 

  25. Jiang, L., Hickman, J. H., Wang, S. J., & Gu, W. (2015). Dynamic roles of p53-mediated metabolic activities in ROS-induced stress responses. Cell Cycle, 14(18), 2881–2885. https://doi.org/10.1080/15384101.2015.1068479

    Article  CAS  Google Scholar 

  26. Yang, W. S., & Stockwell, B. R. (2008). Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells. Chemistry & Biology, 15(3), 234–245. https://doi.org/10.1016/j.chembiol.2008.02.010

    Article  CAS  Google Scholar 

  27. Hadian, K., & Stockwell, B. R. (2020). SnapShot: Ferroptosis. Cell, 181(5), 1188-e1. https://doi.org/10.1016/j.cell.2020.04.039

    Article  CAS  Google Scholar 

  28. Tang, W., Chen, Z., Zhang, W., Cheng, Y., Zhang, B., Wu, F., et al. (2020). The mechanisms of sorafenib resistance in hepatocellular carcinoma: Theoretical basis and therapeutic aspects. Signal Transduction and Targeted Therapy, 5(1), 87. https://doi.org/10.1038/s41392-020-0187-x

    Article  Google Scholar 

  29. Ling, S., Song, L., Fan, N., Feng, T., Liu, L., Yang, X., et al. (2017). Combination of metformin and sorafenib suppresses proliferation and induces autophagy of hepatocellular carcinoma via targeting the mTOR pathway. International Journal of Oncology, 50(1), 297–309. https://doi.org/10.3892/ijo.2016.3799

    Article  CAS  Google Scholar 

  30. Sun, X., Niu, X., Chen, R., He, W., Chen, D., Kang, R., et al. (2016). Metallothionein-1G facilitates sorafenib resistance through inhibition of ferroptosis. Hepatology, 64(2), 488–500. https://doi.org/10.1002/hep.28574

    Article  CAS  Google Scholar 

  31. Nie, J., Lin, B., Zhou, M., Wu, L., & Zheng, T. (2018). Role of ferroptosis in hepatocellular carcinoma. Journal of Cancer Research and Clinical Oncology, 144(12), 2329–37. https://doi.org/10.1007/s00432-018-2740-3

    Article  CAS  Google Scholar 

Download references

Funding

The study was supported by Science and Technology Plan Project of Guangzhou (Grant No. 202002030044) and Fund of Affiliated Huadu Hospital, Southern Medical University (People’s Hospital of Huadu District) (Grant No. 2020B03).

Author information

Author notes

  1. Juan Zhao contributed equally to this work.

Authors and Affiliations

  1. Department of Internal Medicine, Affiliated Huadu Hospital, Southern Medical University (People’s Hospital of Huadu District), Guangzhou, 510800, China

    Hui Li

  2. Department of the Clinical Laboratory, Affiliated Huadu Hospital, Southern Medical University (People’s Hospital of Huadu District), Guangzhou, 510800, China

    Juan Zhao, Li-jun Du, Ping Fang & Mei-juan Li

  3. Gastroenterology of Affiliated Huadu Hospital, Southern Medical University (People’s Hospital of Huadu District), Guangzhou, 510800, China

    Xiao-lan Zhong

  4. Department of Infectious Diseases, Affiliated Huadu Hospital, Southern Medical University (People’s Hospital of Huadu District), Guangzhou, 510800, China

    Pei-yan Xu & Li-juan Tan

  5. Department of Oncology, Affiliated Huadu Hospital, Southern Medical University (People’s Hospital of Huadu District), Guangzhou, 510800, China

    Cheng-fang Zhang

  6. Department of Surgical, Affiliated Huadu Hospital, Southern Medical University (People’s Hospital of Huadu District), Guangzhou, 510800, China

    Tian-sheng Cao

Authors
  1. Hui Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Juan Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xiao-lan Zhong
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Pei-yan Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Li-jun Du
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ping Fang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Li-juan Tan
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Mei-juan Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Cheng-fang Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Tian-sheng Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

(I) Conception and design: J Zhao, T.S. Cao; (II) administrative support: T.S. Cao; (III) provision of study materials or patients: P.Y. Xu, X.L. Zhong; (IV) collection and assembly of data: J. Zhao, H. Li; (V) data analysis and interpretation: L.J. Du, P. Fang, C.F. Zhang; (VI) manuscript writing: all authors; (VII) final approval of manuscript: all authors. H.L. designed the study and revised and edited the manuscript. J.Z. analyzed the data, interpreted the results, and wrote the manuscript. T.S.C. designed and guided the whole experiment. L.J.D., P.F., M.J.L., and C.F.Z. helped with the sample preparation and performed the experiments, and P.Y.X., X.L.Z., and L.J.T. helped with the patients’ clinical assessment and compiled the clinical details. All authors reviewed the manuscript.

Corresponding authors

Correspondence to Juan Zhao or Tian-sheng Cao.

Ethics declarations

Ethics Approval

Our study was approved by the Ethics Committee of the People’s Hospital of Huadu District. All procedures involving people comply with the ethical standards of the institutional and/or national committee for research ethics and the 1964 Helsinki Declaration and its subsequent changes or comparable ethical standards.

Consent to Participate

This article does not contain any studies with human participants performed by any of the authors.

Consent for Publication

All other authors have read the manuscript and have agreed to submit it in its current form for consideration for publication in the Applied Biochemistry and Biotechnology.

Competing Interests

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, H., Zhao, J., Zhong, Xl. et al. CPLX2 Regulates Ferroptosis and Apoptosis Through NRF2 Pathway in Human Hepatocellular Carcinoma Cells. Appl Biochem Biotechnol 195, 597–609 (2023). https://doi.org/10.1007/s12010-022-04135-9

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-022-04135-9

Keywords

Search

Navigation