Pro-caspase-3 protects cells from polymyxin B-induced cytotoxicity by preventing ROS accumulation


Polymyxin B (PMB), a last-line antibiotic used against antibiotic-resistant superbugs, causes undesirable cytotoxic side effects. However, its mechanisms remain unknown. In this study, we unexpectedly found that caspase-3, a main executor of apoptosis, plays a protective role in PMB-induced cytotoxicity. Caspase-3 knockout (KO) cells exhibited higher susceptibility to PMB-induced cytotoxicity compared with wild-type (WT) cells, accompanied by increased levels of reactive oxygen species (ROS). Interestingly, co-treatment with the antioxidant N-acetylcysteine (NAC) rescued cell viability to a similar extent as WT cells. Furthermore, PMB failed to facilitate the processing of inactive caspase-3 (pro-caspase-3) into active forms, suggesting that pro-caspase-3 nonenzymatically suppresses PMB-driven ROS accumulation and its cytotoxicity. Thus, our findings that demonstrate the potential ability of PMB to stimulate ROS generation, but which is normally masked by pro-caspase-3-dependent mechanisms, may provide novel insights into the mechanisms of PMB-induced side effects.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1
Fig. 2


  1. 1.

    Nord NM, Hoeprich PD, Polymyxin B, Colistin. A. Critical Comparison. New Engl J Med. 1964;270:1030–5.

  2. 2.

    Arnold TM, Forrest GN, Messmer KJ. Polymyxin antibiotics for gram-negative infections. Am J Health-Syst Pharm. 2007;64:819–26.

  3. 3.

    Khondker A, Dhaliwal AK, Saem S, Mahmood A, Fradin C, Moran-Mirabal J, et al. Membrane charge and lipid packing determine polymyxin-induced membrane damage. Commun Biol. 2019;2:67.

  4. 4.

    Velkov T, Thompson PE, Nation RL, Li J. Structure–activity relationships of polymyxin antibiotics. J Med Chem. 2010;53:1898–916.

  5. 5.

    Li J, Nation RL. Old polymyxins are back: is resistance close? Clin Infect Dis. 2006;43:663–4.

  6. 6.

    Cai Y, Lee W, Kwa AL. Polymyxin B versus colistin: an update. Expert Rev anti-Infect Ther. 2015;13:1481–97.

  7. 7.

    Kubin CJ, Ellman TM, Phadke V, Haynes LJ, Calfee DP, Yin MT. Incidence and predictors of acute kidney injury associated with intravenous polymyxin B therapy. J Infect. 2012;65:80–87.

  8. 8.

    Azad MA, Finnin BA, Poudyal A, Davis K, Li J, Hill PA, et al. Polymyxin B induces apoptosis in kidney proximal tubular cells. Antimicrob agents Chemother. 2013;57:4329–35.

  9. 9.

    Noguchi T, Tsuchida M, Kogue Y, Spadini C, Hirata Y, Matsuzawa A. Brefeldin A-inhibited guanine nucleotide-exchange factor 1 (BIG1) governs the recruitment of tumor necrosis factor receptor-associated factor 2 (TRAF2) to tumor necrosis factor receptor 1 (TNFR1) signaling complexes. Int J Mol Sci. 2016;17:1869.

  10. 10.

    Hirata Y, Katagiri K, Nagaoka K, Morishita T, Kudoh Y, Hatta T, et al. TRIM48 Promotes ASK1 activation and cell death through ubiquitination-dependent degradation of the ASK1-negative regulator PRMT1. Cell Rep. 2017;21:2447–57.

  11. 11.

    Sekiguchi Y, Yamada M, Noguchi T, Noomote C, Tsuchida M, Kudoh Y, et al. The anti-cancer drug gefitinib accelerates Fas-mediated apoptosis by enhancing caspase-8 activation in cancer cells. J Toxicol Sci. 2019;44:435–40.

  12. 12.

    McArthur K, Kile BT. Apoptotic caspases: multiple or mistaken identities? Trends cell Biol. 2018;28:475–93.

  13. 13.

    Noguchi T, Suzuki M, Mutoh N, Hirata Y, Tsuchida M, Miyagawa S, et al. Nuclear-accumulated SQSTM1/p62-based ALIS act as microdomains sensing cellular stresses and triggering oxidative stress-induced parthanatos. Cell death Dis. 2018;9:1193.

  14. 14.

    Kim JS, Ha JY, Yang SJ, Son JH. A novel non-apoptotic role of procaspase-3 in the regulation of mitochondrial biogenesis activators. J Cell Biochem. 2018;119:347–57.

  15. 15.

    Brentnall M, Weir DB, Rongvaux A, Marcus AI, Boise LH. Procaspase-3 regulates fibronectin secretion and influences adhesion, migration and survival independently of catalytic function. J cell Sci. 2014;127:2217–26.

  16. 16.

    Dezoti Fonseca C, Watanabe M, Vattimo Mde F. Role of heme oxygenase-1 in polymyxin B-induced nephrotoxicity in rats. Antimicrob Agents Chemother. 2012;56:5082–7.

  17. 17.

    Suzuki T, Yamamoto M. Molecular basis of the Keap1-Nrf2 system. Free Radic Biol Med. 2015;88:93–100.

  18. 18.

    Chen M, Ona VO, Li M, Ferrante RJ, Fink KB, Zhu S, et al. Minocycline inhibits caspase-1 and caspase-3 expression and delays mortality in a transgenic mouse model of Huntington disease. Nat Med. 2000;6:797–801.

  19. 19.

    Krajewska M, Wang HG, Krajewski S, Zapata JM, Shabaik A, Gascoyne R, et al. Immunohistochemical analysis of in vivo patterns of expression of CPP32 (caspase-3), a cell death protease. Cancer Res. 1997;57:1605–13.

Download references


This work was supported by JSPS KAKENHI Grant Numbers JP18H02567 and JP18K06622, and by MEXT KAKENHI JP17H05518 and JP19H05282. This work was also supported by the Fugaku Trust for Medicinal Research, the Takeda Science Foundation, and the Division for Interdisciplinary Advanced Research and Education (DIARE) Tohoku University.

Author information



Corresponding authors

Correspondence to Takuya Noguchi or Atsushi Matsuzawa.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Yokosawa, T., Yamada, M., Noguchi, T. et al. Pro-caspase-3 protects cells from polymyxin B-induced cytotoxicity by preventing ROS accumulation. J Antibiot 72, 848–852 (2019).

Download citation

Further reading