Cellular apoptosis: An alternative mechanism of action for caspofungin against Candida glabrata


1 1Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran

2 Department of Immunology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran

3 Infectious and Tropical Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

4 Invasive Fungi Research Centre, Mazandaran University of Medical Sciences, Sari, Iran

5 Department of Medical Laboratory Sciences, Sari Branch, Islamic Azad University, Sari, Iran

6 Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran


Background and Purpose: Although the mechanism of action for echinocandins is known, the physiological mechanisms by which these antifungal agents cause cell death via the classical apoptotic pathways are not well-defined yet. Regarding this, the present study aimed to evaluate the mechanisms of caspofungin-induced Candida glabrata cell death.

Materials and Methods: For the purpose of the study, the minimum inhibitory concentration (MIC) of caspofungin against C. glabrata (ATCC 90030) was determined using the broth microdilution reference method (CLSI M27-A2 and M27-S4). The annexin V and propidium iodide staining was performed to determine the way through which caspofungin acts against C. glabrata (i.e., through the induction of apoptosis and/or necrosis). Additionally, the possible effect of caspofungin on inducing the expression of two apoptotic genes, namely MCA1 and NUC, was studied using the real-time polymerase chain reaction assay.

Results: According to the obtained MIC value (0.5 μg/mL), C. glabrata, exposed to 0.25, 0.5, and 1 μg/mL of caspofungin, exhibited the features of late apoptosis/necrosis after 18 h of incubation. Furthermore, the use of 0.25, 0.5, and 1 μg/ml caspofungin induced apoptosis (early/late) in 14.67%, 17.04%, and 15.89% of the cells, respectively. The results showed a significant difference between the percentages of early-apoptotic cells at the three concentrations (p <0.05). In addition, the rate of necrosis was significantly greater than that of apoptosis in response to caspofungin. Accordingly, necrosis occurred in 71.26%, 71.26%, and 61.26% of the cells at the caspofungin concentrations of 0.25, 0.5, and 1 μg/mL, respectively (p <0.05). The analysis of the data in the REST software demonstrated a significant increase in the expression of MCA1 and NUC1 genes (p <0.05).

Conclusion: As the findings of the present study indicated, caspofungin promoted both necrosis and apoptosis of C. glabrata cells at concentrations higher than or equal to the MIC value.


1. Douglas CM, D'ippolito JA, Shei GJ, Meinz M, Onishi J, Marrinan JA, et al. Identification of the FKS1 gene of Candida albicans as the essential target of 1, 3-beta-D-glucan synthase inhibitors. Antimicrob Agents Chemother. 1997; 41(11):2471-9.
2. Denning DW. Echinocandin antifungal drugs. Lancet. 2003; 362(9390):1142-51.
3. Chandrasekar P, Sobel J. Micafungin: a new echinocandin. Clin Infect Dis. 2006; 42(8):1171-8.
4. Kartsonis NA, Nielsen J, Douglas CM. Caspofungin: the first in a new class of antifungal agents. Drug Resist Updat. 2003; 6(4):197-218.
5. Walker LA, Munro CA, de Bruijn I, Lenardon MD, McKinnon A, Gow NA. Stimulation of chitin synthesis rescues Candida albicans from echinocandins. PLoS Pathog. 2008; 4(4):e1000040.
6. Walker LA, Gow NA, Munro CA. Fungal echinocandin resistance. Fungal Genet Biol. 2010; 47(2):117-26.
7. Barchiesi F, Spreghini E, Tomassetti S, Della Vittoria A, Arzeni D, Manso E, et al. Effects of caspofungin against Candida guilliermondii and Candida parapsilosis. Antimicrob Agents Chemother. 2006; 50(8):2719-27.
8. Pfaller M, Diekema D, Andes D, Arendrup M, Brown S, Lockhart S, et al. Clinical breakpoints for the echinocandins and Candida revisited: integration of molecular, clinical, and microbiological data to arrive at species-specific interpretive criteria. Drug Resist Updat. 2011; 14(3):164-76.
9. Pham CD, Iqbal N, Bolden CB, Kuykendall RJ, Harrison LH, Farley MM, et al. Role of FKS mutations in Candida glabrata: MIC values, echinocandin resistance, and multidrug resistance. Antimicrob Agents Chemother. 2014; 58(8):4690-6.
10. Arendrup MC, Perlin DS. Echinocandin resistance: an emerging clinical problem? Curr Opin Infect Dis. 2014; 27(6):484-92.
11. Aerts AM, Carmona-Gutierrez D, Lefevre S, Govaert G, François IE, Madeo F, et al. The antifungal plant defensin RsAFP2 from radish induces apoptosis in a metacaspase independent way in Candida albicans. FEBS Lett. 2009; 583(15):2513-6.
12. Aerts AM, Bammens L, Govaert G, Carmona-Gutierrez D, Madeo F, Cammue BP, et al. The antifungal plant defensin HsAFP1 from Heuchera sanguinea induces apoptosis in Candida albicans. Front Microbiol. 2011; 2:47.
13. Diaz L, Chiong M, Quest AF, Lavandero S, Stutzin A. Mechanisms of cell death: molecular insights and therapeutic perspectives. Cell Death Differ. 2005; 12(11):1449-56.
14. Collins JA, Schandl CA, Young KK, Vesely J, Willingham MC. Major DNA fragmentation is a late event in apoptosis. J Histochem Cytochem. 1997; 45(7):923-34.
15. Munoz AJ, Wanichthanarak K, Meza E, Petranovic D. Systems biology of yeast cell death. FEMS Yeast Res. 2012; 12(2):249-65.
16. Candé C, Cecconi F, Dessen P, Kroemer G. Apoptosis-inducing factor (AIF): key to the conserved caspase-independent pathways of cell death? J Cell Sci. 2002; 115(24):4727-34.
17. Wu M, Xu LG, Li X, Zhai Z, Shu HB. AMID, an apoptosis-inducing factor-homologous mitochondrion-associated protein, induces caspase-independent apoptosis. J Biol Chem. 2002; 277(28):25617-23.
18. Li LY, Luo X, Wang X. Endonuclease G is an apoptotic DNase when released from mitochondria. Nature. 2001; 412(6842):95-9.
19. Parrish J, Li L, Klotz K, Ledwich D, Wang X, Xue D. Mitochondrial endonuclease G is important for apoptosis in C. elegans. Nature. 2001; 412(6842):90-4.
20. Wayne P. Clinical and Laboratory Standards Institute: reference method for broth dilution antifungal susceptibility testing of yeasts; fourth informational supplement. CLSI Document. 2008; 3:6-12.
21. Clinical and Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of yeasts; fourth informational supplement. Wayne, PA: Clinical and Laboratory Standards Institute; 2012.
22. Almeida B, Sampaio-Marques B, Carvalho J, Silva MT, Leão C, Rodrigues F, et al. An atypical active cell death process underlies the fungicidal activity of ciclopirox olamine against the yeast Saccharomyces cerevisiae. FEMS Yeast Res. 2007; 7(3):404-12.
23. Dobrucki J, Darzynkiewicz Z. Chromatin condensation and sensitivity of DNA in situ to denaturation during cell cycle and apoptosis--a confocal microscopy study. Micron. 2001; 32(7):645-52.
24. Wu XZ, Chang WQ, Cheng AX, Sun LM, Lou HX. Plagiochin E,
an antifungal active macrocyclic bis (bibenzyl), induced apoptosis in Candida albicans through a metacaspase-dependent apoptotic pathway. Biochim Biophys Acta. 2010; 1800(4):439-47.
25. Ligr M, Madeo F, Fröhlich E, Hilt W, Fröhlich KU, Wolf DH. Mammalian Bax triggers apoptotic changes in yeast. FEBS Lett. 1998; 438(1-2):61-5.
26. Degterev A, Boyce M, Yuan J. A decade of caspases. Oncogene. 2003; 22(53):8543-67.
27. Uren AG, O'Rourke K, Aravind L, Pisabarro MT, Seshagiri S, Koonin EV, et al. Identification of paracaspases and metacaspases: two ancient families of caspase-like proteins, one of which plays a key role in MALT lymphoma. Mol Cell. 2000; 6(4):961-7.
28. Madeo F, Herker E, Maldener C, Wissing S, Lächelt S, Herlan M, et al. A caspase-related protease regulates apoptosis in yeast. Mol Cell. 2002; 9(4):911-7.
29. Enari M, Sakahira H, Yokoyama H, Okawa K, Iwamatsu A, Nagata S. A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature. 1998; 391(6662):43-50.
30. Liang Q, Li W, Zhou B. Caspase-independent apoptosis in yeast. Biochim Biophys Acta. 2008; 1783(7):1311-9.
31. Nabili M, Moazeni M, Hedayati MT, Aryamlo P, Gohar AA, Madani SM, et al. Glabridin induces overexpression of two major apoptotic genes, MCA1 and NUC1, in Candida albicans. J Global Antimicrob Resist. 2017; 11:52-6.
32. Moazeni M, Hedayati MT, Nabili M, Mousavi SJ, Abdollahi Gohar A, Gholami S. Glabridin triggers over-expression of MCA1 and NUC1 genes in Candida glabrata: is it an apoptosis inducer? J Mycol Med. 2017; 27(3):369-75.
33. Almeida B, Silva A, Mesquita A, Sampaio-Marques B, Rodrigues F, Ludovico P. Drug-induced apoptosis in yeast. Biochim Biophys Acta. 2008; 1783(7):1436-48.
34. Hwang Is, Lee J, Hwang JH, Kim KJ, Lee DG. Silver nanoparticles induce apoptotic cell death in Candida albicans through the increase of hydroxyl radicals. FEBS J. 2012; 279(7):1327-38.
35. Shirtliff ME, Krom BP, Meijering RA, Peters BM, Zhu J, Scheper MA, et al. Farnesol-induced apoptosis in Candida albicans. Antimicrob Agents Chemother. 2009; 53(6):2392-401.
36. Al-Dhaheri RS, Douglas LJ. Apoptosis in Candida biofilms exposed to amphotericin B. J Med Microbiol. 2010; 59(2):149-57.
37. Hao B, Cheng S, Clancy CJ, Nguyen MH. Caspofungin kills Candida albicans by causing both cellular apoptosis and necrosis. Antimicrob Agents Chemother. 2013; 57(1):326-32.
38. Walker LA, Gow NA, Munro CA. Elevated chitin content reduces the susceptibility of Candida species to caspofungin. Antimicrob Agents Chemother. 2013; 57(1):146-54.