Skip to main content
SpringerLink
Log in

Isoliquiritigenin Induces Cytotoxicity in PC-12 Cells In Vitro

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

Abstract

Isoliquiritigenin (ISL) has been reported to have a wide range of biological activities. This study evaluated the cytotoxic effect of ISL on norvegicus pheochromocytoma cell line (PC-12 cells) and its possible molecular mechanism. The cytotoxicity in vitro of ISL against PC-12 cells was investigated by MTT assay. The migration and invasion of PC-12 cells were performed by scratch test and transwell assay. Apoptosis was evaluated by microscopy and flow cytometry. The reactive oxygen species (ROS) and mitochondrial membrane potential were studied by fluorescent microscopy. DNA damage of PC-12 cells was analyzed by comet assay. The protein expression of caspase, Bcl-2 family member, autophagy-associated protein Beclin-1, and LC3 was detected by western blot. The autophagy of PC-12 cells was investigated by acridine orange (AO) and monodansylcadaverine (MDC) staining. The IC50 value of ISL against PC-12 cell is 17.8 ± 1.8 μM. ISL could suppress PC-12 cell migration and invasion. AO/ethidium bromide staining and flow cytometry suggested that ISL caused apoptosis of PC-12 cells. Significant DNA damages of PC-12 cells treated with ISL were observed in a comet assay. ISL inhibited the cell growth of PC-12 cells at S phase. Exposure of PC-12 cells to ISL increased the levels of cellular reactive oxygen species (ROS) and decreased the mitochondrial membrane potential. Additionally, ISL trigged the release of cytochrome c from the mitochondria to the cytoplasm. The expression levels of caspase-9, caspase-3, caspase-7, Bax, and Bim were upregulated, whereas the expression levels of Bcl-2 and Bcl-x were downregulated. AO and monodansylcadaverine (MDC) staining assay showed that ISL caused autophagy of PC-12 cells. The upregulation of protein Beclin-1 and LC3 was observed in PC-12 cells. Therefore, the results show that ISL induces apoptosis of PC-12 cells through ROS-mediated activation of the intrinsic mitochondria-cytochrome c-caspase protease mechanism and causes the autophagy of PC-12 cells.

The in vitro cytotoxicity, apoptosis, comet assay, ROS, mitochondrial membrane potential, cell cycle arrest, autophagy, and western blot induced by ISL were investigated.

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
Fig. 8
Fig. 9
Fig. 10
Fig. 11
Fig. 12

Similar content being viewed by others

References

  1. Evan, G. I., & Vousden, K. H. (2001). Proliferation cell cycle and apoptosis in cancer. Nature, 41, 342–348.

    Article  Google Scholar 

  2. Fulda, S., & Debatin, K. M. (2004). Signaling through death receptors in cancer therapy. Current Opinion in Pharmacology, 4, 327–332.

    Article  CAS  Google Scholar 

  3. Debatin, K. M., & Krammer, P. H. (2004). Death receptors in chemotherapy and cancer. Oncogene, 23, 2950–2966.

    Article  CAS  Google Scholar 

  4. Hengartner, M. O. (2000). The biochemistry of apoptosis. Nature, 40, 770–776.

    Article  Google Scholar 

  5. Kim, H. E., Du, F., Fang, M., & Wang, X. (2005). Formation of apoptosome is initiated by cytochrome c-induced dATP hydrolysis and subsequent nucleotide exchange on Apaf-1. Proceedings of the National Academy of Sciences of the United States of America, 102, 17545–17550.

    Article  CAS  Google Scholar 

  6. Fang, H. Q., Wu, Y. L., Guo, J. B., Rong, J., Ma, L., Zhao, Z. M., Zuo, D. Y., & Peng, S. Q. (2012). T-2 toxin induces apoptosis in differentiated murine embryonic stem cells through reactive oxygen species-mediated mitochondrial pathway. Apoptosis, 17, 895–907.

    Article  CAS  Google Scholar 

  7. Song, W., Li, S. S., Qiu, P. P., Shen, D. Y., Tian, L., Zhang, Q. Y., Liao, L. X., & Chen, Q. X. (2013). Apoptosis induced by aqueous extracts of crocodile bile in human heptacarcinoma SMMC-7721. Applied Biochemistry and Biotechnology, 170, 15–24.

    Article  CAS  Google Scholar 

  8. Wakx, A., Dutot, M., Massicot, F., Mascarelli, G. F., Limb, A., & Rat, P. (2016). Amyloid β peptide induces apoptosis through P2X7 cell death receptor in retinal cells: modulation by marine omega-3 fatty acid DHA and EPA. Applied Biochemistry and Biotechnology, 178, 368–381.

    Article  CAS  Google Scholar 

  9. Hotchkiss, R. S., Strasser, A., Mc Dunn, J. E., & Swanson, P. E. (2009). Cell death. The New England Journal of Medicine, 361, 1570–1583.

    Article  CAS  Google Scholar 

  10. Tan, C. P., Lai, S. S., Wu, S. H., Hu, S., & Xu, A. L. (2010). Nuclear permeable ruthenium(II) β-carboline complexesinduce autophagyto antagonize mitochondrial mediated apoptosis. Journal of Medicinal Chemistry, 53, 7613–7624.

    Article  CAS  Google Scholar 

  11. Maycotte, P., & Thorburn, A. (2011). Autophagy and cancer therapy. Cancer Biology & Therapy, 11, 127–137.

    Article  CAS  Google Scholar 

  12. Yang, C., Kaushal, V., Shah, S. V., & Kaushal, G. P. (2008). Autophagy is associated with apoptosis in cisplatin injury to renal tubular epithelial cells. American Journal of Physiology Renal, Fluid Electrolyte Physiology, 294, 777–787.

    Article  Google Scholar 

  13. Li, X., Xu, H. L., Liu, Y. X., An, N., Zhao, S., & Bao, J. K. (2013). Autophagy modulation as a target for anticancer drug discovery. Acta Pharmacologica Sinica, 34, 612–624.

    Article  CAS  Google Scholar 

  14. Yuan, L., Wei, S. P., Wang, J., & Liu, X. B. (2014). Isoorientin induces apoptosis and autophagy simultaneously by reactive oxygen species (ROS)-related p53, PI3K/Akt, JNK, and p38 signaling pathways in HepG2 cancer cells. Journal of Agricultural and Food Chemistry, 62, 5390–5400.

    Article  CAS  Google Scholar 

  15. Chen, G., Zhu, L., Liu, Y., Zhou, Q., Chen, H., & Yang, J. (2009). Isoliquiritigenin, a flavonoid from licorice, plays a dual role in regulating gastrointestinal motility in vitro and in vivo. Phytotherapy Research, 23, 498–506.

    Article  CAS  Google Scholar 

  16. Sun, Z. J., Chen, G., Zhang, W., Hu, X., Huang, C. F., Wang, Y. F., Jia, J., & Zhao, Y. F. (2010). Mammalian target of rapamycin pathway promotes tumor-induced angiogenesis in adenoid cystic carcinoma: its suppression by isoliquiritigenin through dual activation of c-Jun NH2-terminal kinase and inhibition of extracellular signalregulated kinase. The Journal of Pharmacology and Experimental Therapeutics, 334, 500–512.

    Article  CAS  Google Scholar 

  17. Park, S. J., Song, H. Y., & Youn, H. S. (2009). Suppression of the TRIF-dependent signaling pathway of toll-like receptors by isoliquiritigenin in RAW264.7 macrophages. Molecular Cell, 28, 365–368.

    Article  CAS  Google Scholar 

  18. Iwashita, K., Kobori, M., Yamaki, K., & Tsushida, T. (2000). Flavonoids inhibit cell growth and induce apoptosis in B16 melanoma 4A5 cells. Bioscience, Biotechnology, and Biochemistry, 64, 1813–1820.

    Article  CAS  Google Scholar 

  19. Chen, X., Yang, M., Hao, W., Han, J., Ma, J., Wang, C., Sun, S., & Zheng, Q. (2016). Differentiation-inducing and anti-proliferative activities of isoliquiritigenin and all-trans-retinoic acid on B16F0 melanoma cells: mechanisms profiling by RNA-seq. Gene, 592, 86–89.

    Article  CAS  Google Scholar 

  20. Wang, Y., Ma, J., Yan, X., Chen, X., Si, L., Liu, Y., Han, J., Hao, W., & Zheng, Q. (2016). Isoliquiritigenin inhibits proliferation and induces apoptosis via alleviating hypoxia and reducing glycolysis in mouse melanoma B16F10 cells. Recent Patents on Anti-Cancer Drug Discovery, 11, 215–217.

    Article  CAS  Google Scholar 

  21. Maggiolini, M., Statti, G., Vivacqua, A., Gabriele, S., Rago, V., Loizzo, M., Menichini, F., & Amdo, S. (2002). Estrogenic and antiproliferative activities of isoliquiritigenin in MCF7 breast cancer cells. The Journal of Steroid Biochemistry and Molecular Biology, 82, 315–322.

    Article  CAS  Google Scholar 

  22. Hsu, Y. L., Kuo, P. L., & Lin, C. C. (2005). Isoliquiritigenin induces apoptosis and cell cycle arrest through p53-dependent pathway in HepG2 cells. Life Sciences, 77, 279–292.

    Article  CAS  Google Scholar 

  23. Hsu, Y. L., Kuo, P. L., Lin, L. T., & Lin, C. C. (2005). Isoliquiritigenin inhibits cell proliferation and induces apoptosis in human hepatoma cells. Planta Medica, 71, 130–134.

    Article  CAS  Google Scholar 

  24. Jung, J. I., Chung, E., Seon, M. R., Shin, H. K., Kim, E. J., Lim, S. S., Chung, W. Y., Park, K. K., & Park, J. H. Y. (2006). Isoliquiritigenin (ISL) inhibits ErbB3 signaling in prostate cancer cells. BioFactors, 28, 159–168.

    Article  CAS  Google Scholar 

  25. Jung, J. I., Lim, S. S., Choi, H. J., Cho, H. J., Shin, H. K., Kim, E. J., Chung, W. Y., Park, K. K., & Park, J. H. Y. (2006). Isoliquiritigen ininduces apoptosis by depolarizing mitochondrial membranes in prostate cancer cells. The Journal of Nutritional Biochemistry, 17, 689–696.

    Article  CAS  Google Scholar 

  26. Zhang, X. Y., Yeung, E. D., Wang, J. Y., Panzhinskiy, E. E., Tong, C., Li, W. G., & Li, J. (2010). Isoliquiritigenin a natural anti-oxidant selectively inhibits the proliferation ofprostate cancer cells. Clinical and Experiment Pharmacology & Physiology, l37, 841–847.

    Google Scholar 

  27. Li, D., Wang, Z., Chen, H., Wang, J., Zheng, Q., Shang, J., & Li, J. (2009). Isoliquiritigenin induces monocytic differentiation of HL-60 cells. Free Radical Biology & Medicine, 46, 731–736.

    Article  Google Scholar 

  28. Ghobrial, I. M., Witzig, T. E., & Adjei, A. A. (2005). Targeting apoptosis pathways in cancertherapy. Cancer Journal for Clinicians, 55, 178–194.

    Article  Google Scholar 

  29. Li, W., Jiang, G. B., Yao, J. H., Wang, X. Z., Wang, J., Han, B. J., Xie, Y. Y., Lin, G. J., Huang, H. L., & Liu, Y. J. (2014). Ruthenium(II) complexes: DNA-binding, cytotoxicity, apoptosis, cellular localization, cell cycle arrest, reactive oxygen species, mitochondrial membrane potential and western blot analysis. Journal of Photochemistry and Photobiology B: Biology, 14, 094–104.

    Article  Google Scholar 

  30. Alapetite, C., Wachter, T., Sage, E., & Moustacchi, E. (1996). Use of the alkaline comet assay to detect DNA repair deficiencies in human fibroblasts exposed to UVC, UVB, UVA and gamma-rays. International Journal of Radiation Biology, 69, 359–369.

    Article  CAS  Google Scholar 

  31. Silveira, L. R., Pereira-Da-Silva, L., Juel, C., & Hellsten, Y. (2003). Formation of hydrogen peroxide and nitric oxide in rat skeletal muscle cells during contractions. Free Radical Biology & Medicine, 35, 455–464.

    Article  CAS  Google Scholar 

  32. Ito, K., Hirao, A., Arai, F., Takubo, K., Matsuoka, S., Miyamoto, K., Ohmura, M., Naka, K., Hosokawa, K., Ikeda, Y., & Suda, T. (2006). Reactive oxygen species act through p38 MAPK to limit the lifespan of hematopoietic stem cells. Nature Medicine, 12, 446–451.

    Article  CAS  Google Scholar 

  33. Chen, H. M., Zhang, B., Yao, Y., Chen, N., Chen, X. Y., Tian, H., Wang, Z. H., & Zheng, Q. S. (2012). NADPH oxidase-derived reactive oxygen species are involved in the HL-60 cell monocytic differentiation induced by isoliquiritigenin. Molecules, 17, 13424–13438.

    Article  CAS  Google Scholar 

  34. Yuan, X., Zhang, B., Chen, N., Chen, X. Y., Liu, L. L., Zheng, Q. S., & Wang, Z. P. (2012). Isoliquiritigenin treatment induces apoptosis by increasing intracellular ROS levels in HeLa cells. Journal of Asian Natural Products Research, 14, 789–798.

    Article  CAS  Google Scholar 

  35. Thress, K., Kornbluth, S., & Smith, J. J. (1999). Mitochondria at the crossroad of apoptotic cell death. Bioenergetics Biomembranes, 31, 321–326.

    Article  CAS  Google Scholar 

  36. Chen, H. M., Zhang, B., Yuan, X., Yao, Y., Zhao, H., Sun, X. L., & Zheng, Q. S. (2013). Isoliquiritigenin-induced effects on Nrf2 mediated antioxidant defence in the HL-60 cell monocytic differentiation. Cell Biology International, 37, 1215–1224.

    CAS  Google Scholar 

  37. Maiuri, M. C., Zalckvar, E., Kimchi, A., & Kroemer, G. (2007). Self-eating and self-killing: crosstalk between autophagy and apoptosis. Molecular and Cellular Biology, 8, 741–745.

    CAS  Google Scholar 

  38. Song, W., Yang, H. B., Chen, P., Wang, S. M., Zhao, L. P., Xu, W. H., Fan, H. F., Gu, X., & Chen, L. Y. (2013). Apoptosis of human gastric carcinoma SGC-7901 induced by deoxycholic acid via the mitochondrial-dependent pathway. Applied Biochemistry and Biotechnology, 171, 1061–1071.

    Article  CAS  Google Scholar 

  39. Takahashi, T., Takasuka, N., Iigo, M., Baba, M., Nishino, H., Tsuda, H., & Okuyama, T. (2004). Isoliquiritigenin, a flavonoid from licorice, reduces prostaglandin E2 and nitric oxide, causes apoptosis, andsuppresses aberrant crypt foci development. Cancer Science, 95, 448–453.

    Article  CAS  Google Scholar 

  40. Chen, G., Hu, X., Zhang, W., Xu, N., Wang, F. Q., Jia, J., Zhang, W. F., Sun, Z. J., & Zhao, Y. F. (2012). Mammalian target of rapamycin regulates isoliquiritigenin induced autophagic and apoptotic cell death in adenoid cystic carcinoma cells. Apoptosis, 17, 90–101.

    Article  CAS  Google Scholar 

  41. Madamanchi, N. R., Moon, S. K., Hakim, Z. S., Clark, S., Mehrizi, A., Patterson, C., & Runge, M. S. (2005). Differential activation of mitogenic signaling pathways in aortic smooth muscle cells deficient in superoxide dismutase isoforms. Arteriosclerosis, Thrombosis, and Vascular Biology, 25, 950–956.

    Article  CAS  Google Scholar 

  42. Poli, G. (2004). Oxidative stress and cell signalling. Current Medicinal Chemistry, 11, 1163–1182.

    Article  CAS  Google Scholar 

  43. Yuan, X., Zhang, B., Gan, L., Wang, Z. H., Yu, B. C., Liu, L. L., Zheng, Q. S., & Wang, Z. P. (2013). Involvement of the mitochondrion-dependent and the endoplasmic reticulum stress-signaling pathways in isoliquiritigenin-induced apoptosis of HeLa cell. Biomedical and Environmental Sciences, 26, 268–276.

    CAS  Google Scholar 

  44. Singh, N. P. (1988). A simple technique for quantification of low levels of DNA damage in individual cells. Experimental Cell Research, 175, 184–191.

    Article  CAS  Google Scholar 

  45. Orrenius, S., Gogvadze, V., & Zhivotovsky, B. (2007). Mitochondrial oxidative stress: implications for cell death. Annual Review of Pharmacology and Toxicology, 47, 143–183.

    Article  CAS  Google Scholar 

  46. Li, P. (1997). Cytochrome c and dATP-dependent formation ofApaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell, 91, 479–489.

    Article  CAS  Google Scholar 

  47. Kaufmann, S. H., & Hengartner, M. O. (2001). Programmed cell death: alive and well in the new millennium. Trends in Cell Biology, 11, 526–534.

    Article  CAS  Google Scholar 

  48. Arthur, C. R., Gupton, J. T., Kellogg, G. E., Yeudall, W. A., Cabot, W. C., Newsham, I. F., & Gewirtz, D. A. (2007). Autophagic cell death, polyploidy and senescence induced inbreast tumor cells by the substituted pyrrole JG-03-14, a novelmicrotubule poison. Biochemical Pharmacology, 74, 981–991.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the Priority Academic Program Development of Guangdong Higher Education Institution (2013LYM0047), the Natural Science Foundation of Guangdong Province (No. 2016A030313728) and the project of innovation for enhancing Guangdong Pharmceutical University, provincial experimental teaching demonstration center of chemistry and chemical engineering.

Author information

Authors and Affiliations

  1. School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, 510006, People’s Republic of China

    Hui-Hui Yang, Cheng Zhang, Shang-Hai Lai, Chuan-Chuan Zeng, Yun-Jun Liu & Xiu-Zhen Wang

  2. Guangdong Cosmetics Engineering and Technology Research Center, Guangzhou, 510006, People’s Republic of China

    Yun-Jun Liu

Authors
  1. Hui-Hui Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Cheng Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shang-Hai Lai
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chuan-Chuan Zeng
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yun-Jun Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Xiu-Zhen Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Yun-Jun Liu or Xiu-Zhen Wang.

Additional information

Research Highlights

• The cytotoxicity of ISL was evaluated by MTT method.

• The apoptosis was investigated with AO/EB and flow cytometry.

• The cycle arrest, ROS, and mitochondrial membrane potential were studied.

• Investigation of the expression of proteins in apoptosis pathway was performed.

• The autophagy and autophagy-associated proteins were analyzed.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yang, HH., Zhang, C., Lai, SH. et al. Isoliquiritigenin Induces Cytotoxicity in PC-12 Cells In Vitro. Appl Biochem Biotechnol 183, 1173–1190 (2017). https://doi.org/10.1007/s12010-017-2491-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-017-2491-7

Keywords

Search

Navigation