- Email: [email protected]
β-lapachone and α-nor-lapachone modulate Candida albicans viability and virulence factors
G Model
MYCMED-785; No. of Pages 6 Journal de Mycologie Me´dicale xxx (2018) xxx–xxx
Available online at
ScienceDirect www.sciencedirect.com
Original article/Article original
b-lapachone and a-nor-lapachone modulate Candida albicans viability and virulence factors D.C. Moraes a,*, J.A.R. Curvelo a, C.A. Anjos a, K.C.G. Moura b, M.C.F.R. Pinto b, M.B. Portela c, R.M.A. Soares a a b c
Instituto de Microbiologia Paulo de Go´es, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil Instituto de Pesquisa de Produtos Naturais, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil Departamento de Clı´nica e Odontopediatria, Universidade Federal Fluminense, Rio de Janeiro, Brasil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 November 2017 Received in revised form 5 March 2018 Accepted 9 March 2018 Available online xxx
Background. – Candida albicans is the most important fungal pathogen that causes infections in humans, and the search for new therapeutic strategies for its treatment is essential. Objective. – The aim of this study was to evaluate the activity of seven naphthoquinones (b-lapachone, b-nor-lapachone, bromide-b-lapachone, hydroxy-b-lapachone, a-lapachone, a-nor-lapachone and axyloidone) on the growth of a fluconazole-resistant C. albicans oral clinical isolate and the effects of these compounds on the viability of mammalian cells, on yeast’s morphogenesis, biofilm formation and cell wall mannoproteins availability. Results. – All the compounds were able to completely inhibit the yeast growth. b-lapachone and a-norlapachone were the less cytotoxic compounds against L929 and RAW 264.7 cells. At IC50, b-lapachone inhibited morphogenesis in 92%, while the treatment of yeast cells with a-nor-lapachone decreased yeast-to-hyphae transition in 42%. At 50 mg/ml, b-lapachone inhibited biofilm formation by 84%, whereas a-nor-lapachone reduced biofilm formation by 64%. The treatment of yeast cells with blapachone decreased cell wall mannoproteins availability in 28.5%, while a-nor-lapachone was not able to interfere on this virulence factor. Taken together, data show that b-lapachone and a-nor-lapachone exhibited in vitro cytotoxicity against a fluconazole-resistant C. albicans strain, thus demonstrating to be promising candidates to be used in the treatment of infections caused by this fungus.
C 2018 Elsevier Masson SAS. All rights reserved.
Keywords: Candida albicans Lapachone Antifungal activity Biofilm Fluconazole resistance
1. Introduction Candida albicans is the most important fungal pathogen that causes infections in humans [1]. Nevertheless, the research for new therapeutic strategies for its treatment continues to be essential due to the high mortality associated with candidiasis, especially in immunocompromised individuals, as well as the small number of drugs available for treatment, the adverse effects associated with the usage of certain compounds and the increasing incidence of antifungal resistance [2]. Since fungal and animal cells share a conserved eukaryotic biology, the discovery of new antifungal medicines has been considered an outstanding challenge to modern research [3]. An alternative strategy for the development of anti-C. albicans drugs may be the use of virulence factors of yeast as pharmacological
* Corresponding author. Instituto de Microbiologia Paulo de Go´es, Centro de Cieˆncias da Sau´de, Universidade Federal do Rio de Janeiro, Bloco I, Cidade Universita´ria, 21941-590, Rio de Janeiro, Brasil. E-mail address: [email protected] (D.C. Moraes).
targets. These virulence factors can be defined as features that allow the establishment of infection, and hence, of the disease itself [4]. C. albicans has developed several of these virulence factors throughout its evolution, and three of the most relevant of them are the cell wall mannoprotein production [5], the yeast-tohyphae transition, also called morphogenesis [6] and the biofilm formation [7]. Natural products have been traditionally used as therapeutic agents. In the last two decades, several studies have found that quinone compounds exhibit valuable pharmacological properties such as antitumor [8] and trypanocidal activities [9]. The development of synthetic organic chemistry has allowed structural modifications of compounds obtained naturally, in order to improve pharmacological and/or toxicological properties. Two examples are b-lapachone and a-nor-lapachone, quinones that possess a variety of pharmacological activities and can be synthesized from lapachol [10,11]. Due to the current epidemiological situation of candidiasis and the difficulty in obtaining effective therapeutic strategies, mainly against infections caused by resistant microorganisms, the aim of
https://doi.org/10.1016/j.mycmed.2018.03.004 C 2018 Elsevier Masson SAS. All rights reserved. 1156-5233/
Please cite this article in press as: Moraes DC, et al. b-lapachone and a-nor-lapachone modulate Candida albicans viability and virulence factors. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.03.004
G Model
MYCMED-785; No. of Pages 6 D.C. Moraes et al. / Journal de Mycologie Me´dicale xxx (2018) xxx–xxx
2
A C. albicans strain (namely PRI) isolated from the oral mucosa of a pediatric HIV patient known to be resistant to fluconazole (MIC > 256 mg/ml) was used in this study [12]. Yeasts were maintained at low temperature (liquid nitrogen) and before any experimental procedures, cells were subcultured into brain heart infusion (BHI) broth and incubated at 37 8C for 48 h to reach exponential growing phase.
were used in this study, L929 fibroblast cells and RAW 264.7 macrophage cells obtained from mice. Serial dilutions of the compounds were made, using Minimum Eagle Medium (MEM) (Cultilab1, Sa˜o Paulo, Brazil) without serum as diluent. Then, the substances were placed in contact with confluent cell monolayers. The cells were incubated at 37 8C for 72 h in 5% CO2. Then, neutral red dye was added to each well of a 96-well flat-bottomed microtitre plate, followed by incubation at 37 8C for 3 h. After this period, the dye was discarded; the cell monolayers were washed three times with PBS 10 mM (KH2PO4 0.43 g, NaCl 7.2 g, Na2HPO4 2.54 g, final volume 1000 ml, pH adjusted to 7.2) and added 4% formaldehyde during 5 minutes for fixation. After this procedure, a solution containing 1% acetic acid and 50% methanol was added to the system. Cells were then incubated at room temperature and the cell viability was evaluated in a spectrophotometer (FLUOstar OPTIMA, BMG Labtech1, Offenburg, Germany) at 492 nm. The results are expressed as the concentration of compounds able of inhibiting mitochondrial metabolism by 50%, also referred as CC50.
2.2. Compounds
2.5. Morphogenesis
b-lapachone (b-lap), b-nor-lapachone (b-nor-lap), bromide-blapachone (Br-b-lap), hydroxy-b-lapachone (OH-b-lap), a-lapachone (a-lap), a-nor-lapachone (a-nor-lap) and a-xyloidone (axyl) (Fig. 1) were synthesized by Laboratory of Heterocyclic Chemistry from Institute of Natural Products Research (IPPN/ UFRJ). Compounds were dissolved in dimethyl sulfoxyde (Sigma Aldrich1, St. Louis, USA) to a final concentration of 10 mg/ml.
The effect of the naphtoquinones on yeast-to-hyphae transition was evaluated as described by Braga-Silva and coworkers (2007) with slight modifications. Briefly, 5 106 cells were incubated in 1.0 ml fetal bovine serum (FCS) (Cultilab1, Sa˜o Paulo, Brazil) in the presence or absence of the compounds at IC50 for 3 h at 37 8C with agitation (75 rpm). Cell viability was tested by the trypan blue exclusion method. Then, cells were suspended in 1 M NaOH containing 10 mM EDTA and 1% (v/v) b-mercaptoethanol to eliminate clusters that could have hampered counting accuracy. A haemocytometer Neubauer chamber was used for differential counting, and the percentage of germ tubes was determined. This procedure was performed four times, and a minimum of 500 cells were counted at each experiment.
this study was to evaluate the activity of seven naphthoquinones on the growth of a fluconazole-resistant C. albicans oral clinical isolate, and the effects of these compounds on the viability of mammalian cells, on yeast’s morphogenesis, biofilm formation and cell wall mannoproteins availability.
2. Materials and methods 2.1. Yeast strain and growth conditions
2.3. Antifungal susceptibility testing The minimal inhibitory concentration (MIC) was determined according to the M27-A3 methodology for microdilution from CLSI [13]. Briefly, cells were inoculated into RPMI-1640 medium at a concentration of 5 103 cells/ml and incubated at 37 8C for 48 hours with agitation (75 rpm), in the presence of serial concentrations (100–0.2 mg/ml) of the compounds. Cell growth was measured using a microplate reader at 600 nm (Fluostar Optima, BMG Labtech, Offenburg, Germany). The minimum concentration able to inhibit 100% and 50% of yeast growth were defined as MIC and IC50, respectively. 2.4. Cytotoxicity Cytotoxicity of the compounds was determined using the ‘‘dyeuptake’’ technique [14] with slight modifications. Two cell lines
2.6. Biofilm formation inhibition The effect of the naphthoquinones on biofilm formation was evaluated as described by Thein and coworkers (2007) with slight modifications [15]. Briefly, 106 cells were added to a 96-well flatbottomed microtitre plate and incubated at 37 8C for 90 min with gentle agitation (75 rpm). Supernatant was removed and wells were gently washed twice with PBS to remove non-adherent cells. YNB medium BD1 (Franklin NJ, USA) supplemented with 100 mM of glucose containing serial concentrations of the compounds (range: 100–0.2 mg/ml) was added to the plates containing the
Fig. 1. Structure of the compounds tested in this study. 1–b-lapachone; 2–b-nor-lapachone; 3–bromide-b-lapachone; 4–hydroxy-b-lapachone; 5–a-lapachone; 6–a-norlapachone; 7–a-xyloidone.
Please cite this article in press as: Moraes DC, et al. b-lapachone and a-nor-lapachone modulate Candida albicans viability and virulence factors. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.03.004
G Model
MYCMED-785; No. of Pages 6 D.C. Moraes et al. / Journal de Mycologie Me´dicale xxx (2018) xxx–xxx
cells and incubated at 37 8C for 48 h. After incubation, supernatant was removed and cells were gently washed twice in PBS to remove non-adherent cells. Biofilm quantification was performed by MTT reduction assay, and metabolic activity of the cells was measured spectrophotometrically at 492 nm (Fluostar Optima, BMG Labtech, Germany). 2.7. Cell wall mannoproteins availability In order to evaluate variations in the availability of mannoproteins on the cell wall, flow cytometry analysis was carried out [16]. Briefly, 1 107 cells were incubated with IC50 of the naphthoquinones for 6 h at 37 8C. After incubation, cells were washed with PBS 10 mM, fixed in paraformaldehyde 4%, blocked with BSA 5% and incubated for 1 h with FITC labelled Concanavalin-A (Sigma Aldrich1 St. Louis, USA) 2 mg/ml in a 1:500 dilution. Cells were washed and 10.000 events were acquired for each analysis in an EPICS ELITE1 flow cytometer (Coulter Electronics, Hialeah, FL, USA) equipped with a 15 mW argon laser at a wavelength of 488 nm. 2.8. Statistical analysis All experiments were performed at least three times and the results were expressed as mean standard deviation. Data were analyzed by Student’s t-test, and P-values lower than 0.05 were considered significant.
3
Table 2 CC50 of the seven naphthoquinones against L929 cell line (fibroblasts) and RAW 264.7 cell line (macrophages) from Mus musculus. Cell growth was evaluated by neutral red dye-uptake and measured spectrophotometrically (492 nm). Compound
CC50
b-lapachone b-nor-lapachone Bromide-b-lapachone Hydroxy-b-lapachone a-lapachone a-nor-lapachone a-xyloidone
L929 (mg/ml) 8.88 4.78 < 3.10 < 3.10 < 3.10 42.97 < 3.10
RAW 264.7 (mg/ml) 9.57 5.02 < 3.10 < 3.10 < 3.10 34.00 < 3.10
lower cytotoxic activities to both mammal cells, they were chosen for the remaining experiments. 3.3. Morphogenesis Fig. 2 shows the effect of subinhibitory concentrations of a-norlap and b-lap against morphogenesis of PRI strain. Dimethyl sulfoxide at 1% v/v was used as control, since it was used to solubilize the compounds. Treatment of cells with a-nor-lap 0.06 mg/ml led to a decrease of 42% in germ tube differentiation, in comparison to the control. Moreover, the treatment with b-lap 0.77 mg/ml inhibited morphogenesis in 92%. Dimethyl sulfoxide did not affect germ tube differentiation significantly. 3.4. Biofilm formation inhibition
3. Results 3.1. Antifungal susceptibility testing MIC and IC50 values are summarized in Table 1. Results show that all the seven naphthoquinones were able to completely inhibit yeast growth, in a range concentration from 1.33 mg/ml (a-norlap) to 73.98 mg/ml (a-lap). 3.2. Cytotoxicity CC50 values for the seven naphthoquinones against murine fibroblasts and macrophages are summarized in Table 2. Results obtained show that a-nor-lap was the less toxic compound against both L929 and RAW264.7 cells, presenting CC50 values of 42.97 mg/ ml and 34.00 mg/ml, respectively. b-lap and b-nor-lap also presented satisfactory CC50 values related to their MIC. Their CC50 to L929 and RAW 264.7 cells were 8.88 mg/ml and 9.57 mg/ml for b-lap and 4.78 mg/ml and 5.02 mg/ml for b-nor-lap, respectively, and the other four compounds completely inhibited the growth of L929 and RAW 264.7 cell lines at the lowest tested concentration. Considering the results obtained up to now, and the fact that a-nor-lap and b-lap presented the best MIC values and
The effect of a-nor-lap and b-lap on PRI biofilm formation is shown in Fig. 3. Data reveal that both compounds inhibit biofilm formation in a dose-dependent manner. At 25 mg/ml, no significant difference was detected between biofilm formation inhibition by the compounds. However, at higher concentrations, b-lap presented a greater antibiofilm activity than a-nor-lap. At 50 mg/ ml, b-lap inhibited biofilm formation by 84%, whereas a-nor-lap reduced biofilm formation by 64%. At 100 mg/ml, the highest tested concentration, the biofilm formation inhibition by b-lap reached 92%, and the treatment with a-nor-lap led to 82% of inhibition. 3.5. Cell wall mannoproteins availability Fig. 4 shows that the treatment of PRI strain with the IC50 of blap decreased the fluorescence emitted by conA-FITC in 28.5% (inset). However, when cells were incubated with a-nor-lap, fluorescence emitted by the fluorescent probe did not change
Table 1 MIC and IC50 of seven naphthoquinones assessed on a fluconazole-resistant C. albicans clinical isolate, according to the CLSI M27-A3 protocol. Cell growth was measured spectrophotometrically (600 nm). Compound
MIC (mg/ml)
IC50 (mg/ml)
b-lapachone b-nor-lapachone Bromide-b-lapachone Hydroxy-b-lapachone a-lapachone a-nor-lapachone a-xyloidone
2.76 0.09 20.02 0.21 24.33 0.01 35.42 2.19 73.98 23.58 1.33 0.01 50.21 1.26
0.77 0.63 4.49 0.83 7.80 0.50 11.19 2.27 39.39 12.26 0.06 0.04 20.04 1.37
Fig. 2. Effect of a-nor-lapachone and b-lapachone on yeast-to-hyphae transition (morphogenesis) of a fluconazole-resistant C. albicans clinical isolate (PRI strain). Myceliation was calculated by differential counting under optical microscopy. * Statistically significant difference between treated systems and control system (P < 0.05).
Please cite this article in press as: Moraes DC, et al. b-lapachone and a-nor-lapachone modulate Candida albicans viability and virulence factors. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.03.004
G Model
MYCMED-785; No. of Pages 6 D.C. Moraes et al. / Journal de Mycologie Me´dicale xxx (2018) xxx–xxx
4
Fig. 3. Effect of serial concentrations of a-nor-lap and b-lap on the biofilm formation process of C. albicans (PRI strain). YNB medium containing a-nor-lap or b-lap was placed on adhered cells and incubated at 37 8C for 48 h, and quantification was performed by MTT assay reduction. *P = 0.05, and the result is statistically significant between each compound and the untreated system. ** P = 0.05, and the result is statistically significant between b-lap and a-nor-lap.
(Fig. 4b and inset). Altogether, these results point that b-lap somehow decreases the availability of mannoproteins on C. albicans cell wall surface, while a-nor-lap does not present this capability. 4. Discussion In this study, it was firstly evaluated the influence of seven lapachone-derivative compounds on the growth of a fluconazoleresistant C. albicans oral clinical isolate. Results show that all the
tested compounds were able to inhibit fungal growth in some degree. Considering the b-lap derivatives, it can be observed that b-lap itself has presented the lowest MIC value, and the substitution of a hydrogen atom of the b-lap C-ring by a bromide atom or a hydroxyl group led to a large increase of MIC value. It could be explained either by the charge distribution across the carbon-bromide and carbon-oxygen bonds, since the carbon atom is less electronegative than the other two atoms, or by the increase of molecular weight. Interestingly, b-nor-lap has presented a MIC value comparable to Br-b-lap. Therefore, molecular weight may not be a major feature to explain the difference observed on the MIC values. Amongst the a-lap derivatives, a-nor-lap has presented a MIC value about 50-fold lesser than a-xyl, and 70-fold lesser than alap. It may be explained by the fact that a-xyl and a-lap C-ring have one methylene group more than a-nor-lap. A striking feature of quinoidal compounds is their ability to induce the production of reactive oxygen species (ROS), such as OH., O2. and H2O2 and, according to Ramos-Pe´rez and coworkers (2014), b-lap exerts a cytotoxic effect on yeasts due to a massive ROS production, which promotes lipid peroxidation, damage on proteins and nucleic acids and DNA fragmentation [17]. Although all tested compounds were able to induce ROS production, the kinetics of this process depends on the reduction potential of each quinone, and this parameter may be affected by the presence of substituents on C-ring [18]. Since any quinone is likely to undergo bioreduction and generate ROS, the assessment of the cytotoxicity of the compounds used in this study against mammalian cells is highly relevant. It was observed that all b-lap and a-lap derivatives were cytotoxic against RAW cells and L929 cells, but CC50 values for b-lap and a-
Fig. 4. Analysis by flow cytometry showing binding of conA-FITC to the surface of C. albicans (PRI strain). Yeasts were treated with b-lap (a) or a-nor-lap (b) and incubated for 6 h at 37 8C. Then, cells were incubated for 1 h in presence of conA labelled with FITC. Autofluorescence was given by incubating the cells in the absence of conA-FITC (black lines). Treated cells are represented by the blue lines, while untreated cells are represented by red lines. The arbitrary fluorescence units (A.F.U.) were quantified and are represented by the inset figure. *Statistically significant difference between b-lap system and control system (P < 0.05).
Please cite this article in press as: Moraes DC, et al. b-lapachone and a-nor-lapachone modulate Candida albicans viability and virulence factors. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.03.004
G Model
MYCMED-785; No. of Pages 6 D.C. Moraes et al. / Journal de Mycologie Me´dicale xxx (2018) xxx–xxx
nor-lap were higher than their MIC values. Moreover, it was observed that a-nor-lap has presented higher antifungal activity than the b-lap, but was also less cytotoxic against the two cell lines used in the cytotoxicity assay of this study. Taken together, these results suggest that there are other mechanisms involved in the pharmacological activities of naphthoquinones besides ROS production. If only the oxidative stress were responsible for the biological properties of quinoidal compounds, it would be natural to expect that the compound with the highest antifungal activity would also be the one with the highest cytotoxicity to mammalian cells. The ability of switching to filamentous forms provides C. albicans a greater adherence on the epithelial surface of host tissue, when compared with the yeast form [19]. Furthermore, filamentous form can exert mechanical force, favoring epithelial penetration and endothelial damage, which can aid the escape of the microorganism from the bloodstream into deeper tissues, drastically worsening prognosis [20]. This study revealed a significant ability of naphthoquinones of inhibiting yeast-tohyphae transition. At 0.77 mg/ml, b-lap has almost completely prevented the dimorphism process, while a-nor-lap at 0.06 mg/ml reduced the process by 42%. Several studies have been done in order to identify the function of low molecular weight molecules in the modulation of dimorphism process. For instance, it is known that substances such as farnesol, cyclooxygenase inhibitors and propranolol regulate this process through different mechanisms, as interfering at the mitogen-activated protein (MAP) kinase pathway, or by decreasing Ras1p concentration [21,22]. Moreover, Tsang, Bandara and Fong (2012) observed that purpurin, an anthraquinone, suppressed the development of hyphae and the formation of C. albicans biofilm by decreasing the expression of hyphae-specific genes (ALS3, ECE1, HWP1, HYR1 and RAS1) [23]. Biofilm formation by C. albicans is considered an important virulence factor, since this structure is able to develop on medical devices such as central venous catheters, urinary catheters, artificial heart valves, among others, enabling the spread of the microorganism into the bloodstream [24]. Moreover, biofilmgrown C. albicans cells show greater antifungal resistance than planktonic cells, making it difficult to eradicate this structure by drug treatments [7]. At the highest concentration tested (100 mg/ ml), b-lap and a-nor-lap have reduced biofilm formation by 92% and 82%, respectively. This concentration is thirty-six-fold higher than b-lap MIC, and seventy-five-fold higher than a-nor-lap MIC, corroborating with the affirmative that biofilms exhibit increased resistance to antifungal agents. However, b-lap and a-nor-lap were able to significantly affect biofilm formation at a low concentration (1.56 mg/ml), pointing that both the compounds are interesting tools to be explored as antibiofilm agents. Several mechanisms are related to antimicrobial resistance of biofilms. There may be mentioned, for example, slow diffusion of the substance through the biofilm; chemical changes in the biofilm microenvironment; adaptive responses to stress; presence of a small population of tolerant cells; expression of efflux pumps, among others [25]. The activity of b-lap and a-nor-lap on biofilm formation in C. albicans may be associated with the inhibition of morphogenesis by these substances, since hyphae development has an important role in the composition of the upper biofilm layer [26]. This association reinforces the effective ability of both compounds in modulating a so important and refractory virulence factor such as fungal biofilm [4]. The cell wall mannoproteins of C. albicans, or adhesins, play an important role in the interaction between fungus and host cells, since these structures confer adherence to biotic and abiotic surfaces [27]. Therefore, substances able to decrease the expression or activity of these glycoconjugates would reduce the potential of the species to promote its pathogenesis, and may be
5
recognized as extremely relevant in prognosis improvement. In this study, it was observed that the treatment of fungal cells with b-lap reduced the binding of concanavalin-A to the cell wall mannoproteins by 28.5%. However, the treatment with a-nor-lap was not able to modify the availability of these structures. These results suggest that the interaction between the compounds and C. albicans cell wall mannoproteins may be related to the difference in carbonyl positioning of the lapachone isomers. Since cell wall mannoproteins are required in biofilm formation [28], the effect of b-lap on this process in C. albicans could be explained by the modulation of these adhesins. In summary, data obtained in this study suggest that blapachone and a-nor-lapachone are promising candidates to be used in the treatment of infections caused by C. albicans, since these quinones showed great antifungal activity at safe concentrations and were able to inhibit yeast-to-hyphae transition, biofilm formation and cell wall mannoproteins availability, three of the most important virulence factors associated to the development and prognosis of C. albicans infections. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Disclosure of interest The authors declare that they have no competing interest. References [1] Wille MP, Guimara˜es T, Campos Furtado GH, Colombo AL. Historical trends in the epidemiology of candidaemia: analysis of an 11-year period in a tertiary care hospital in Brazil. Mem Inst Oswaldo Cruz 2013;108:288–92. http:// dx.doi.org/10.1590/0074-0276108032013005. [2] Kathiravan MK, Salake AB, Chothe AS, Dudhe PB, Watode RP, Mukta MS, et al. The biology and chemistry of antifungal agents: a review. Bioorganic Med Chem 2012;20:5678–98. http://dx.doi.org/10.1016/j.bmc.2012.04.045. [3] Gulshan K, Moye-Rowley WS. Multidrug resistance in fungi. Eukaryot Cell 2007;6:1933–42. http://dx.doi.org/10.1128/EC.00254-07. [4] Mayer FL, Wilson D, Hube B. Candida albicans pathogenicity mechanisms. Virulence 2013;4:119–28. http://dx.doi.org/10.4161/viru.22913. [5] Gow NAR, Hube B. Importance of the Candida albicans cell wall during commensalism and infection. Curr Opin Microbiol 2012;15:406–12. http:// dx.doi.org/10.1016/j.mib.2012.04.005. [6] Gow NAR, Brown AJP, Odds FC. Fungal morphogenesis and host invasion. Curr Opin Microbiol 2002;5:366–71. http://dx.doi.org/10.1016/S13695274(02)00338-7. [7] Fox EP, Nobile CJ. A sticky situation: untangling the transcriptional network controlling biofilm development in Candida albicans. Transcription 2012;3:315–22. http://dx.doi.org/10.4161/trns.22281. [8] Da Cruz EHG, Silvers MA, Jardim GAM, Resende JM, Cavalcanti BC, Bomfim IS, et al. Synthesis and antitumor activity of selenium-containing quinone-based triazoles possessing two redox centres, and their mechanistic insights. Eur J Med Chem 2016;122:1–16. http://dx.doi.org/10.1016/j.ejmech.2016.06.019. [9] Suto Y, Nakajima-Shimada J, Yamagiwa N, Onizuka Y, Iwasaki G. Synthesis and biological evaluation of quinones derived from natural product komaroviquinone as anti-Trypanosoma cruzi agents. Bioorganic Med Chem Lett 2015;25:2967–71. http://dx.doi.org/10.1016/j.bmcl.2015.05.022. [10] Rı´os-Luci C, Bonifazi EL, Leo´n LG, Montero JC, Burton G, Pandiella A, et al. bLapachone analogs with enhanced antiproliferative activity. Eur J Med Chem 2012;53:264–74. http://dx.doi.org/10.1016/j.ejmech.2012.04.008. [11] Tseng CH, Cheng CM, Tzeng CC, Peng SI, Yang CL, Chen YL. Synthesis and antiinflammatory evaluations of b-lapachone derivatives. Bioorganic Med Chem 2013;21:523–31. http://dx.doi.org/10.1016/j.bmc.2012.10.047. [12] Braga-Silva LA, Souza Dos Santos AL, Portela MB, Souto-Padro´n T, Soares RMDA. Effect of suramin on the human pathogen Candida albicans: implications on the fungal development and virulence. FEMS Immunol Med Microbiol 2007;51:399–406. http://dx.doi.org/10.1111/j.1574-695X.2007.00321.x. [13] Rex JH, Alexander BD, Andes D, Arthington-Skaggs B, Brown SD, Chaturvedi V, et al. Reference method for broth dilution antifungal susceptibility testing of yeasts: approved standard - third edition. Clin Lab Stand Inst 2008;1–25. [14] Neyndorff HC, Bartel DL, Tufaro F, Levy JG. Development of a model to demonstrate photosensitizer-mediated viral inactivation in blood. Transfusion 1990;30:485–90. [15] Thein ZM, Samaranayake YH, Samaranayake LP. In vitro biofilm formation of Candida albicans and non-albicans Candida species under dynamic and
Please cite this article in press as: Moraes DC, et al. b-lapachone and a-nor-lapachone modulate Candida albicans viability and virulence factors. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.03.004
G Model
MYCMED-785; No. of Pages 6 D.C. Moraes et al. / Journal de Mycologie Me´dicale xxx (2018) xxx–xxx
6
[16]
[17]
[18]
[19]
[20]
anaerobic conditions. Arch Oral Biol 2007;52:761–7. http://dx.doi.org/ 10.1016/j.archoralbio.2007.01.009. Curvelo JADR, Barreto ALS, Portela MB, Alviano DS, Holandino C, Souto-Padro´n T, et al. Effect of the secretory leucocyte proteinase inhibitor (SLPI) on Candida albicans biological processes: a therapeutic alternative? Arch Oral Biol 2014;59:928–37. http://dx.doi.org/10.1016/j.archoralbio.2014.05.007. Ramos-Pe´rez C, Lorenzo-Castrillejo I, Quevedo O, Garcı´a-Luis J, MatosPerdomo E, Medina-Coello C, et al. Yeast cytotoxic sensitivity to the antitumour agent b-lapachone depends mainly on oxidative stress and is largely independent of microtubule- or topoisomerase-mediated DNA damage. Biochem Pharmacol 2014;92:206–19. http://dx.doi.org/10.1016/ j.bcp.2014.09.006. dos Santos EV, Carneiro JW, Ferreira VF. Quantitative structure-activity relationship in aziridinyl-1,4-naphthoquinone antimalarials: study of theoretical correlations by the PM3 method. Bioorganic Med Chem 2004;12:87–93. http://dx.doi.org/10.1016/j.bmc.2003.10.022. Kimura LH, Pearsall NN. Relationship between germination of Candida albicans and increased adherence to human buccal epithelial cells. Infect Immun 1980;28:464–8. Wa¨chtler B, Wilson D, Haedicke K, Dalle F, Hibe B. From attachment to damage: defined genes of Candida albicans mediate adhesion, invasion and damage during interaction with oral epithelial cells. PLoS One 2011;6:e17046. http://dx.doi.org/10.1371/journal.pone.0017046.
[21] Toenjes KA, Munsee SM, Ibrahim AS, Jeffrey R, Jr JEE, Johnson DI, et al. Smallmolecule inhibitors of the budded-to-hyphal-form transition in the pathogenic yeast Candida albicans small-molecule inhibitors of the budded-tohyphal-form transition in the pathogenic yeast Candida albicans. Antimicrob Agents Chemother 2005;49:963–72. http://dx.doi.org/10.1128/AAC.49.3.963. [22] Shareck J, Belhumeur P. Modulation of morphogenesis in Candida albicans by various small molecules. Eukaryot Cell 2011;10:1004–12. http://dx.doi.org/ 10.1128/EC.05030-11. [23] Tsang PWK, Bandara HMHN, Fong WP. Purpurin suppresses Candida albicans biofilm formation and hyphal development. PLoS One 2012;7:1–8. http:// dx.doi.org/10.1371/journal.pone.0050866. [24] Donlan RM. Biofilms and device-associated infections. Emerg Infect Dis 2001;7:277–81. http://dx.doi.org/10.3201/eid0702.010226. [25] Van Acker H, Van Dijck P, Coenye T. Molecular mechanisms of antimicrobial tolerance and resistance in bacterial and fungal biofilms. Trends Microbiol 2014;22:326–33. http://dx.doi.org/10.1016/j.tim.2014.02.001. [26] Soll DR, Daniels KJ. Plasticity of Candida albicans biofilms. Microbiol Mol Biol Rev 2016;80:565–95. http://dx.doi.org/10.1128/MMBR.00068-15. [27] Modrzewska B, Kurnatowski P. Adherence of Candida sp. to host tissues and cells as one of its pathogenicity features. Ann Parasitol 2015;61:3–9. [28] ten Cate JM, Klis FM, Pereira-Cenci T, Crielaard W, de Groot PWJ. Molecular and cellular mechanisms that lead to Candida biofilm formation. J Dent Res 2009;88:105–15. http://dx.doi.org/10.1177/0022034508329273.
Please cite this article in press as: Moraes DC, et al. b-lapachone and a-nor-lapachone modulate Candida albicans viability and virulence factors. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.03.004
MYCMED-785; No. of Pages 6 Journal de Mycologie Me´dicale xxx (2018) xxx–xxx
Available online at
ScienceDirect www.sciencedirect.com
Original article/Article original
b-lapachone and a-nor-lapachone modulate Candida albicans viability and virulence factors D.C. Moraes a,*, J.A.R. Curvelo a, C.A. Anjos a, K.C.G. Moura b, M.C.F.R. Pinto b, M.B. Portela c, R.M.A. Soares a a b c
Instituto de Microbiologia Paulo de Go´es, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil Instituto de Pesquisa de Produtos Naturais, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil Departamento de Clı´nica e Odontopediatria, Universidade Federal Fluminense, Rio de Janeiro, Brasil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 November 2017 Received in revised form 5 March 2018 Accepted 9 March 2018 Available online xxx
Background. – Candida albicans is the most important fungal pathogen that causes infections in humans, and the search for new therapeutic strategies for its treatment is essential. Objective. – The aim of this study was to evaluate the activity of seven naphthoquinones (b-lapachone, b-nor-lapachone, bromide-b-lapachone, hydroxy-b-lapachone, a-lapachone, a-nor-lapachone and axyloidone) on the growth of a fluconazole-resistant C. albicans oral clinical isolate and the effects of these compounds on the viability of mammalian cells, on yeast’s morphogenesis, biofilm formation and cell wall mannoproteins availability. Results. – All the compounds were able to completely inhibit the yeast growth. b-lapachone and a-norlapachone were the less cytotoxic compounds against L929 and RAW 264.7 cells. At IC50, b-lapachone inhibited morphogenesis in 92%, while the treatment of yeast cells with a-nor-lapachone decreased yeast-to-hyphae transition in 42%. At 50 mg/ml, b-lapachone inhibited biofilm formation by 84%, whereas a-nor-lapachone reduced biofilm formation by 64%. The treatment of yeast cells with blapachone decreased cell wall mannoproteins availability in 28.5%, while a-nor-lapachone was not able to interfere on this virulence factor. Taken together, data show that b-lapachone and a-nor-lapachone exhibited in vitro cytotoxicity against a fluconazole-resistant C. albicans strain, thus demonstrating to be promising candidates to be used in the treatment of infections caused by this fungus.
C 2018 Elsevier Masson SAS. All rights reserved.
Keywords: Candida albicans Lapachone Antifungal activity Biofilm Fluconazole resistance
1. Introduction Candida albicans is the most important fungal pathogen that causes infections in humans [1]. Nevertheless, the research for new therapeutic strategies for its treatment continues to be essential due to the high mortality associated with candidiasis, especially in immunocompromised individuals, as well as the small number of drugs available for treatment, the adverse effects associated with the usage of certain compounds and the increasing incidence of antifungal resistance [2]. Since fungal and animal cells share a conserved eukaryotic biology, the discovery of new antifungal medicines has been considered an outstanding challenge to modern research [3]. An alternative strategy for the development of anti-C. albicans drugs may be the use of virulence factors of yeast as pharmacological
* Corresponding author. Instituto de Microbiologia Paulo de Go´es, Centro de Cieˆncias da Sau´de, Universidade Federal do Rio de Janeiro, Bloco I, Cidade Universita´ria, 21941-590, Rio de Janeiro, Brasil. E-mail address: [email protected] (D.C. Moraes).
targets. These virulence factors can be defined as features that allow the establishment of infection, and hence, of the disease itself [4]. C. albicans has developed several of these virulence factors throughout its evolution, and three of the most relevant of them are the cell wall mannoprotein production [5], the yeast-tohyphae transition, also called morphogenesis [6] and the biofilm formation [7]. Natural products have been traditionally used as therapeutic agents. In the last two decades, several studies have found that quinone compounds exhibit valuable pharmacological properties such as antitumor [8] and trypanocidal activities [9]. The development of synthetic organic chemistry has allowed structural modifications of compounds obtained naturally, in order to improve pharmacological and/or toxicological properties. Two examples are b-lapachone and a-nor-lapachone, quinones that possess a variety of pharmacological activities and can be synthesized from lapachol [10,11]. Due to the current epidemiological situation of candidiasis and the difficulty in obtaining effective therapeutic strategies, mainly against infections caused by resistant microorganisms, the aim of
https://doi.org/10.1016/j.mycmed.2018.03.004 C 2018 Elsevier Masson SAS. All rights reserved. 1156-5233/
Please cite this article in press as: Moraes DC, et al. b-lapachone and a-nor-lapachone modulate Candida albicans viability and virulence factors. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.03.004
G Model
MYCMED-785; No. of Pages 6 D.C. Moraes et al. / Journal de Mycologie Me´dicale xxx (2018) xxx–xxx
2
A C. albicans strain (namely PRI) isolated from the oral mucosa of a pediatric HIV patient known to be resistant to fluconazole (MIC > 256 mg/ml) was used in this study [12]. Yeasts were maintained at low temperature (liquid nitrogen) and before any experimental procedures, cells were subcultured into brain heart infusion (BHI) broth and incubated at 37 8C for 48 h to reach exponential growing phase.
were used in this study, L929 fibroblast cells and RAW 264.7 macrophage cells obtained from mice. Serial dilutions of the compounds were made, using Minimum Eagle Medium (MEM) (Cultilab1, Sa˜o Paulo, Brazil) without serum as diluent. Then, the substances were placed in contact with confluent cell monolayers. The cells were incubated at 37 8C for 72 h in 5% CO2. Then, neutral red dye was added to each well of a 96-well flat-bottomed microtitre plate, followed by incubation at 37 8C for 3 h. After this period, the dye was discarded; the cell monolayers were washed three times with PBS 10 mM (KH2PO4 0.43 g, NaCl 7.2 g, Na2HPO4 2.54 g, final volume 1000 ml, pH adjusted to 7.2) and added 4% formaldehyde during 5 minutes for fixation. After this procedure, a solution containing 1% acetic acid and 50% methanol was added to the system. Cells were then incubated at room temperature and the cell viability was evaluated in a spectrophotometer (FLUOstar OPTIMA, BMG Labtech1, Offenburg, Germany) at 492 nm. The results are expressed as the concentration of compounds able of inhibiting mitochondrial metabolism by 50%, also referred as CC50.
2.2. Compounds
2.5. Morphogenesis
b-lapachone (b-lap), b-nor-lapachone (b-nor-lap), bromide-blapachone (Br-b-lap), hydroxy-b-lapachone (OH-b-lap), a-lapachone (a-lap), a-nor-lapachone (a-nor-lap) and a-xyloidone (axyl) (Fig. 1) were synthesized by Laboratory of Heterocyclic Chemistry from Institute of Natural Products Research (IPPN/ UFRJ). Compounds were dissolved in dimethyl sulfoxyde (Sigma Aldrich1, St. Louis, USA) to a final concentration of 10 mg/ml.
The effect of the naphtoquinones on yeast-to-hyphae transition was evaluated as described by Braga-Silva and coworkers (2007) with slight modifications. Briefly, 5 106 cells were incubated in 1.0 ml fetal bovine serum (FCS) (Cultilab1, Sa˜o Paulo, Brazil) in the presence or absence of the compounds at IC50 for 3 h at 37 8C with agitation (75 rpm). Cell viability was tested by the trypan blue exclusion method. Then, cells were suspended in 1 M NaOH containing 10 mM EDTA and 1% (v/v) b-mercaptoethanol to eliminate clusters that could have hampered counting accuracy. A haemocytometer Neubauer chamber was used for differential counting, and the percentage of germ tubes was determined. This procedure was performed four times, and a minimum of 500 cells were counted at each experiment.
this study was to evaluate the activity of seven naphthoquinones on the growth of a fluconazole-resistant C. albicans oral clinical isolate, and the effects of these compounds on the viability of mammalian cells, on yeast’s morphogenesis, biofilm formation and cell wall mannoproteins availability.
2. Materials and methods 2.1. Yeast strain and growth conditions
2.3. Antifungal susceptibility testing The minimal inhibitory concentration (MIC) was determined according to the M27-A3 methodology for microdilution from CLSI [13]. Briefly, cells were inoculated into RPMI-1640 medium at a concentration of 5 103 cells/ml and incubated at 37 8C for 48 hours with agitation (75 rpm), in the presence of serial concentrations (100–0.2 mg/ml) of the compounds. Cell growth was measured using a microplate reader at 600 nm (Fluostar Optima, BMG Labtech, Offenburg, Germany). The minimum concentration able to inhibit 100% and 50% of yeast growth were defined as MIC and IC50, respectively. 2.4. Cytotoxicity Cytotoxicity of the compounds was determined using the ‘‘dyeuptake’’ technique [14] with slight modifications. Two cell lines
2.6. Biofilm formation inhibition The effect of the naphthoquinones on biofilm formation was evaluated as described by Thein and coworkers (2007) with slight modifications [15]. Briefly, 106 cells were added to a 96-well flatbottomed microtitre plate and incubated at 37 8C for 90 min with gentle agitation (75 rpm). Supernatant was removed and wells were gently washed twice with PBS to remove non-adherent cells. YNB medium BD1 (Franklin NJ, USA) supplemented with 100 mM of glucose containing serial concentrations of the compounds (range: 100–0.2 mg/ml) was added to the plates containing the
Fig. 1. Structure of the compounds tested in this study. 1–b-lapachone; 2–b-nor-lapachone; 3–bromide-b-lapachone; 4–hydroxy-b-lapachone; 5–a-lapachone; 6–a-norlapachone; 7–a-xyloidone.
Please cite this article in press as: Moraes DC, et al. b-lapachone and a-nor-lapachone modulate Candida albicans viability and virulence factors. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.03.004
G Model
MYCMED-785; No. of Pages 6 D.C. Moraes et al. / Journal de Mycologie Me´dicale xxx (2018) xxx–xxx
cells and incubated at 37 8C for 48 h. After incubation, supernatant was removed and cells were gently washed twice in PBS to remove non-adherent cells. Biofilm quantification was performed by MTT reduction assay, and metabolic activity of the cells was measured spectrophotometrically at 492 nm (Fluostar Optima, BMG Labtech, Germany). 2.7. Cell wall mannoproteins availability In order to evaluate variations in the availability of mannoproteins on the cell wall, flow cytometry analysis was carried out [16]. Briefly, 1 107 cells were incubated with IC50 of the naphthoquinones for 6 h at 37 8C. After incubation, cells were washed with PBS 10 mM, fixed in paraformaldehyde 4%, blocked with BSA 5% and incubated for 1 h with FITC labelled Concanavalin-A (Sigma Aldrich1 St. Louis, USA) 2 mg/ml in a 1:500 dilution. Cells were washed and 10.000 events were acquired for each analysis in an EPICS ELITE1 flow cytometer (Coulter Electronics, Hialeah, FL, USA) equipped with a 15 mW argon laser at a wavelength of 488 nm. 2.8. Statistical analysis All experiments were performed at least three times and the results were expressed as mean standard deviation. Data were analyzed by Student’s t-test, and P-values lower than 0.05 were considered significant.
3
Table 2 CC50 of the seven naphthoquinones against L929 cell line (fibroblasts) and RAW 264.7 cell line (macrophages) from Mus musculus. Cell growth was evaluated by neutral red dye-uptake and measured spectrophotometrically (492 nm). Compound
CC50
b-lapachone b-nor-lapachone Bromide-b-lapachone Hydroxy-b-lapachone a-lapachone a-nor-lapachone a-xyloidone
L929 (mg/ml) 8.88 4.78 < 3.10 < 3.10 < 3.10 42.97 < 3.10
RAW 264.7 (mg/ml) 9.57 5.02 < 3.10 < 3.10 < 3.10 34.00 < 3.10
lower cytotoxic activities to both mammal cells, they were chosen for the remaining experiments. 3.3. Morphogenesis Fig. 2 shows the effect of subinhibitory concentrations of a-norlap and b-lap against morphogenesis of PRI strain. Dimethyl sulfoxide at 1% v/v was used as control, since it was used to solubilize the compounds. Treatment of cells with a-nor-lap 0.06 mg/ml led to a decrease of 42% in germ tube differentiation, in comparison to the control. Moreover, the treatment with b-lap 0.77 mg/ml inhibited morphogenesis in 92%. Dimethyl sulfoxide did not affect germ tube differentiation significantly. 3.4. Biofilm formation inhibition
3. Results 3.1. Antifungal susceptibility testing MIC and IC50 values are summarized in Table 1. Results show that all the seven naphthoquinones were able to completely inhibit yeast growth, in a range concentration from 1.33 mg/ml (a-norlap) to 73.98 mg/ml (a-lap). 3.2. Cytotoxicity CC50 values for the seven naphthoquinones against murine fibroblasts and macrophages are summarized in Table 2. Results obtained show that a-nor-lap was the less toxic compound against both L929 and RAW264.7 cells, presenting CC50 values of 42.97 mg/ ml and 34.00 mg/ml, respectively. b-lap and b-nor-lap also presented satisfactory CC50 values related to their MIC. Their CC50 to L929 and RAW 264.7 cells were 8.88 mg/ml and 9.57 mg/ml for b-lap and 4.78 mg/ml and 5.02 mg/ml for b-nor-lap, respectively, and the other four compounds completely inhibited the growth of L929 and RAW 264.7 cell lines at the lowest tested concentration. Considering the results obtained up to now, and the fact that a-nor-lap and b-lap presented the best MIC values and
The effect of a-nor-lap and b-lap on PRI biofilm formation is shown in Fig. 3. Data reveal that both compounds inhibit biofilm formation in a dose-dependent manner. At 25 mg/ml, no significant difference was detected between biofilm formation inhibition by the compounds. However, at higher concentrations, b-lap presented a greater antibiofilm activity than a-nor-lap. At 50 mg/ ml, b-lap inhibited biofilm formation by 84%, whereas a-nor-lap reduced biofilm formation by 64%. At 100 mg/ml, the highest tested concentration, the biofilm formation inhibition by b-lap reached 92%, and the treatment with a-nor-lap led to 82% of inhibition. 3.5. Cell wall mannoproteins availability Fig. 4 shows that the treatment of PRI strain with the IC50 of blap decreased the fluorescence emitted by conA-FITC in 28.5% (inset). However, when cells were incubated with a-nor-lap, fluorescence emitted by the fluorescent probe did not change
Table 1 MIC and IC50 of seven naphthoquinones assessed on a fluconazole-resistant C. albicans clinical isolate, according to the CLSI M27-A3 protocol. Cell growth was measured spectrophotometrically (600 nm). Compound
MIC (mg/ml)
IC50 (mg/ml)
b-lapachone b-nor-lapachone Bromide-b-lapachone Hydroxy-b-lapachone a-lapachone a-nor-lapachone a-xyloidone
2.76 0.09 20.02 0.21 24.33 0.01 35.42 2.19 73.98 23.58 1.33 0.01 50.21 1.26
0.77 0.63 4.49 0.83 7.80 0.50 11.19 2.27 39.39 12.26 0.06 0.04 20.04 1.37
Fig. 2. Effect of a-nor-lapachone and b-lapachone on yeast-to-hyphae transition (morphogenesis) of a fluconazole-resistant C. albicans clinical isolate (PRI strain). Myceliation was calculated by differential counting under optical microscopy. * Statistically significant difference between treated systems and control system (P < 0.05).
Please cite this article in press as: Moraes DC, et al. b-lapachone and a-nor-lapachone modulate Candida albicans viability and virulence factors. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.03.004
G Model
MYCMED-785; No. of Pages 6 D.C. Moraes et al. / Journal de Mycologie Me´dicale xxx (2018) xxx–xxx
4
Fig. 3. Effect of serial concentrations of a-nor-lap and b-lap on the biofilm formation process of C. albicans (PRI strain). YNB medium containing a-nor-lap or b-lap was placed on adhered cells and incubated at 37 8C for 48 h, and quantification was performed by MTT assay reduction. *P = 0.05, and the result is statistically significant between each compound and the untreated system. ** P = 0.05, and the result is statistically significant between b-lap and a-nor-lap.
(Fig. 4b and inset). Altogether, these results point that b-lap somehow decreases the availability of mannoproteins on C. albicans cell wall surface, while a-nor-lap does not present this capability. 4. Discussion In this study, it was firstly evaluated the influence of seven lapachone-derivative compounds on the growth of a fluconazoleresistant C. albicans oral clinical isolate. Results show that all the
tested compounds were able to inhibit fungal growth in some degree. Considering the b-lap derivatives, it can be observed that b-lap itself has presented the lowest MIC value, and the substitution of a hydrogen atom of the b-lap C-ring by a bromide atom or a hydroxyl group led to a large increase of MIC value. It could be explained either by the charge distribution across the carbon-bromide and carbon-oxygen bonds, since the carbon atom is less electronegative than the other two atoms, or by the increase of molecular weight. Interestingly, b-nor-lap has presented a MIC value comparable to Br-b-lap. Therefore, molecular weight may not be a major feature to explain the difference observed on the MIC values. Amongst the a-lap derivatives, a-nor-lap has presented a MIC value about 50-fold lesser than a-xyl, and 70-fold lesser than alap. It may be explained by the fact that a-xyl and a-lap C-ring have one methylene group more than a-nor-lap. A striking feature of quinoidal compounds is their ability to induce the production of reactive oxygen species (ROS), such as OH., O2. and H2O2 and, according to Ramos-Pe´rez and coworkers (2014), b-lap exerts a cytotoxic effect on yeasts due to a massive ROS production, which promotes lipid peroxidation, damage on proteins and nucleic acids and DNA fragmentation [17]. Although all tested compounds were able to induce ROS production, the kinetics of this process depends on the reduction potential of each quinone, and this parameter may be affected by the presence of substituents on C-ring [18]. Since any quinone is likely to undergo bioreduction and generate ROS, the assessment of the cytotoxicity of the compounds used in this study against mammalian cells is highly relevant. It was observed that all b-lap and a-lap derivatives were cytotoxic against RAW cells and L929 cells, but CC50 values for b-lap and a-
Fig. 4. Analysis by flow cytometry showing binding of conA-FITC to the surface of C. albicans (PRI strain). Yeasts were treated with b-lap (a) or a-nor-lap (b) and incubated for 6 h at 37 8C. Then, cells were incubated for 1 h in presence of conA labelled with FITC. Autofluorescence was given by incubating the cells in the absence of conA-FITC (black lines). Treated cells are represented by the blue lines, while untreated cells are represented by red lines. The arbitrary fluorescence units (A.F.U.) were quantified and are represented by the inset figure. *Statistically significant difference between b-lap system and control system (P < 0.05).
Please cite this article in press as: Moraes DC, et al. b-lapachone and a-nor-lapachone modulate Candida albicans viability and virulence factors. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.03.004
G Model
MYCMED-785; No. of Pages 6 D.C. Moraes et al. / Journal de Mycologie Me´dicale xxx (2018) xxx–xxx
nor-lap were higher than their MIC values. Moreover, it was observed that a-nor-lap has presented higher antifungal activity than the b-lap, but was also less cytotoxic against the two cell lines used in the cytotoxicity assay of this study. Taken together, these results suggest that there are other mechanisms involved in the pharmacological activities of naphthoquinones besides ROS production. If only the oxidative stress were responsible for the biological properties of quinoidal compounds, it would be natural to expect that the compound with the highest antifungal activity would also be the one with the highest cytotoxicity to mammalian cells. The ability of switching to filamentous forms provides C. albicans a greater adherence on the epithelial surface of host tissue, when compared with the yeast form [19]. Furthermore, filamentous form can exert mechanical force, favoring epithelial penetration and endothelial damage, which can aid the escape of the microorganism from the bloodstream into deeper tissues, drastically worsening prognosis [20]. This study revealed a significant ability of naphthoquinones of inhibiting yeast-tohyphae transition. At 0.77 mg/ml, b-lap has almost completely prevented the dimorphism process, while a-nor-lap at 0.06 mg/ml reduced the process by 42%. Several studies have been done in order to identify the function of low molecular weight molecules in the modulation of dimorphism process. For instance, it is known that substances such as farnesol, cyclooxygenase inhibitors and propranolol regulate this process through different mechanisms, as interfering at the mitogen-activated protein (MAP) kinase pathway, or by decreasing Ras1p concentration [21,22]. Moreover, Tsang, Bandara and Fong (2012) observed that purpurin, an anthraquinone, suppressed the development of hyphae and the formation of C. albicans biofilm by decreasing the expression of hyphae-specific genes (ALS3, ECE1, HWP1, HYR1 and RAS1) [23]. Biofilm formation by C. albicans is considered an important virulence factor, since this structure is able to develop on medical devices such as central venous catheters, urinary catheters, artificial heart valves, among others, enabling the spread of the microorganism into the bloodstream [24]. Moreover, biofilmgrown C. albicans cells show greater antifungal resistance than planktonic cells, making it difficult to eradicate this structure by drug treatments [7]. At the highest concentration tested (100 mg/ ml), b-lap and a-nor-lap have reduced biofilm formation by 92% and 82%, respectively. This concentration is thirty-six-fold higher than b-lap MIC, and seventy-five-fold higher than a-nor-lap MIC, corroborating with the affirmative that biofilms exhibit increased resistance to antifungal agents. However, b-lap and a-nor-lap were able to significantly affect biofilm formation at a low concentration (1.56 mg/ml), pointing that both the compounds are interesting tools to be explored as antibiofilm agents. Several mechanisms are related to antimicrobial resistance of biofilms. There may be mentioned, for example, slow diffusion of the substance through the biofilm; chemical changes in the biofilm microenvironment; adaptive responses to stress; presence of a small population of tolerant cells; expression of efflux pumps, among others [25]. The activity of b-lap and a-nor-lap on biofilm formation in C. albicans may be associated with the inhibition of morphogenesis by these substances, since hyphae development has an important role in the composition of the upper biofilm layer [26]. This association reinforces the effective ability of both compounds in modulating a so important and refractory virulence factor such as fungal biofilm [4]. The cell wall mannoproteins of C. albicans, or adhesins, play an important role in the interaction between fungus and host cells, since these structures confer adherence to biotic and abiotic surfaces [27]. Therefore, substances able to decrease the expression or activity of these glycoconjugates would reduce the potential of the species to promote its pathogenesis, and may be
5
recognized as extremely relevant in prognosis improvement. In this study, it was observed that the treatment of fungal cells with b-lap reduced the binding of concanavalin-A to the cell wall mannoproteins by 28.5%. However, the treatment with a-nor-lap was not able to modify the availability of these structures. These results suggest that the interaction between the compounds and C. albicans cell wall mannoproteins may be related to the difference in carbonyl positioning of the lapachone isomers. Since cell wall mannoproteins are required in biofilm formation [28], the effect of b-lap on this process in C. albicans could be explained by the modulation of these adhesins. In summary, data obtained in this study suggest that blapachone and a-nor-lapachone are promising candidates to be used in the treatment of infections caused by C. albicans, since these quinones showed great antifungal activity at safe concentrations and were able to inhibit yeast-to-hyphae transition, biofilm formation and cell wall mannoproteins availability, three of the most important virulence factors associated to the development and prognosis of C. albicans infections. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Disclosure of interest The authors declare that they have no competing interest. References [1] Wille MP, Guimara˜es T, Campos Furtado GH, Colombo AL. Historical trends in the epidemiology of candidaemia: analysis of an 11-year period in a tertiary care hospital in Brazil. Mem Inst Oswaldo Cruz 2013;108:288–92. http:// dx.doi.org/10.1590/0074-0276108032013005. [2] Kathiravan MK, Salake AB, Chothe AS, Dudhe PB, Watode RP, Mukta MS, et al. The biology and chemistry of antifungal agents: a review. Bioorganic Med Chem 2012;20:5678–98. http://dx.doi.org/10.1016/j.bmc.2012.04.045. [3] Gulshan K, Moye-Rowley WS. Multidrug resistance in fungi. Eukaryot Cell 2007;6:1933–42. http://dx.doi.org/10.1128/EC.00254-07. [4] Mayer FL, Wilson D, Hube B. Candida albicans pathogenicity mechanisms. Virulence 2013;4:119–28. http://dx.doi.org/10.4161/viru.22913. [5] Gow NAR, Hube B. Importance of the Candida albicans cell wall during commensalism and infection. Curr Opin Microbiol 2012;15:406–12. http:// dx.doi.org/10.1016/j.mib.2012.04.005. [6] Gow NAR, Brown AJP, Odds FC. Fungal morphogenesis and host invasion. Curr Opin Microbiol 2002;5:366–71. http://dx.doi.org/10.1016/S13695274(02)00338-7. [7] Fox EP, Nobile CJ. A sticky situation: untangling the transcriptional network controlling biofilm development in Candida albicans. Transcription 2012;3:315–22. http://dx.doi.org/10.4161/trns.22281. [8] Da Cruz EHG, Silvers MA, Jardim GAM, Resende JM, Cavalcanti BC, Bomfim IS, et al. Synthesis and antitumor activity of selenium-containing quinone-based triazoles possessing two redox centres, and their mechanistic insights. Eur J Med Chem 2016;122:1–16. http://dx.doi.org/10.1016/j.ejmech.2016.06.019. [9] Suto Y, Nakajima-Shimada J, Yamagiwa N, Onizuka Y, Iwasaki G. Synthesis and biological evaluation of quinones derived from natural product komaroviquinone as anti-Trypanosoma cruzi agents. Bioorganic Med Chem Lett 2015;25:2967–71. http://dx.doi.org/10.1016/j.bmcl.2015.05.022. [10] Rı´os-Luci C, Bonifazi EL, Leo´n LG, Montero JC, Burton G, Pandiella A, et al. bLapachone analogs with enhanced antiproliferative activity. Eur J Med Chem 2012;53:264–74. http://dx.doi.org/10.1016/j.ejmech.2012.04.008. [11] Tseng CH, Cheng CM, Tzeng CC, Peng SI, Yang CL, Chen YL. Synthesis and antiinflammatory evaluations of b-lapachone derivatives. Bioorganic Med Chem 2013;21:523–31. http://dx.doi.org/10.1016/j.bmc.2012.10.047. [12] Braga-Silva LA, Souza Dos Santos AL, Portela MB, Souto-Padro´n T, Soares RMDA. Effect of suramin on the human pathogen Candida albicans: implications on the fungal development and virulence. FEMS Immunol Med Microbiol 2007;51:399–406. http://dx.doi.org/10.1111/j.1574-695X.2007.00321.x. [13] Rex JH, Alexander BD, Andes D, Arthington-Skaggs B, Brown SD, Chaturvedi V, et al. Reference method for broth dilution antifungal susceptibility testing of yeasts: approved standard - third edition. Clin Lab Stand Inst 2008;1–25. [14] Neyndorff HC, Bartel DL, Tufaro F, Levy JG. Development of a model to demonstrate photosensitizer-mediated viral inactivation in blood. Transfusion 1990;30:485–90. [15] Thein ZM, Samaranayake YH, Samaranayake LP. In vitro biofilm formation of Candida albicans and non-albicans Candida species under dynamic and
Please cite this article in press as: Moraes DC, et al. b-lapachone and a-nor-lapachone modulate Candida albicans viability and virulence factors. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.03.004
G Model
MYCMED-785; No. of Pages 6 D.C. Moraes et al. / Journal de Mycologie Me´dicale xxx (2018) xxx–xxx
6
[16]
[17]
[18]
[19]
[20]
anaerobic conditions. Arch Oral Biol 2007;52:761–7. http://dx.doi.org/ 10.1016/j.archoralbio.2007.01.009. Curvelo JADR, Barreto ALS, Portela MB, Alviano DS, Holandino C, Souto-Padro´n T, et al. Effect of the secretory leucocyte proteinase inhibitor (SLPI) on Candida albicans biological processes: a therapeutic alternative? Arch Oral Biol 2014;59:928–37. http://dx.doi.org/10.1016/j.archoralbio.2014.05.007. Ramos-Pe´rez C, Lorenzo-Castrillejo I, Quevedo O, Garcı´a-Luis J, MatosPerdomo E, Medina-Coello C, et al. Yeast cytotoxic sensitivity to the antitumour agent b-lapachone depends mainly on oxidative stress and is largely independent of microtubule- or topoisomerase-mediated DNA damage. Biochem Pharmacol 2014;92:206–19. http://dx.doi.org/10.1016/ j.bcp.2014.09.006. dos Santos EV, Carneiro JW, Ferreira VF. Quantitative structure-activity relationship in aziridinyl-1,4-naphthoquinone antimalarials: study of theoretical correlations by the PM3 method. Bioorganic Med Chem 2004;12:87–93. http://dx.doi.org/10.1016/j.bmc.2003.10.022. Kimura LH, Pearsall NN. Relationship between germination of Candida albicans and increased adherence to human buccal epithelial cells. Infect Immun 1980;28:464–8. Wa¨chtler B, Wilson D, Haedicke K, Dalle F, Hibe B. From attachment to damage: defined genes of Candida albicans mediate adhesion, invasion and damage during interaction with oral epithelial cells. PLoS One 2011;6:e17046. http://dx.doi.org/10.1371/journal.pone.0017046.
[21] Toenjes KA, Munsee SM, Ibrahim AS, Jeffrey R, Jr JEE, Johnson DI, et al. Smallmolecule inhibitors of the budded-to-hyphal-form transition in the pathogenic yeast Candida albicans small-molecule inhibitors of the budded-tohyphal-form transition in the pathogenic yeast Candida albicans. Antimicrob Agents Chemother 2005;49:963–72. http://dx.doi.org/10.1128/AAC.49.3.963. [22] Shareck J, Belhumeur P. Modulation of morphogenesis in Candida albicans by various small molecules. Eukaryot Cell 2011;10:1004–12. http://dx.doi.org/ 10.1128/EC.05030-11. [23] Tsang PWK, Bandara HMHN, Fong WP. Purpurin suppresses Candida albicans biofilm formation and hyphal development. PLoS One 2012;7:1–8. http:// dx.doi.org/10.1371/journal.pone.0050866. [24] Donlan RM. Biofilms and device-associated infections. Emerg Infect Dis 2001;7:277–81. http://dx.doi.org/10.3201/eid0702.010226. [25] Van Acker H, Van Dijck P, Coenye T. Molecular mechanisms of antimicrobial tolerance and resistance in bacterial and fungal biofilms. Trends Microbiol 2014;22:326–33. http://dx.doi.org/10.1016/j.tim.2014.02.001. [26] Soll DR, Daniels KJ. Plasticity of Candida albicans biofilms. Microbiol Mol Biol Rev 2016;80:565–95. http://dx.doi.org/10.1128/MMBR.00068-15. [27] Modrzewska B, Kurnatowski P. Adherence of Candida sp. to host tissues and cells as one of its pathogenicity features. Ann Parasitol 2015;61:3–9. [28] ten Cate JM, Klis FM, Pereira-Cenci T, Crielaard W, de Groot PWJ. Molecular and cellular mechanisms that lead to Candida biofilm formation. J Dent Res 2009;88:105–15. http://dx.doi.org/10.1177/0022034508329273.
Please cite this article in press as: Moraes DC, et al. b-lapachone and a-nor-lapachone modulate Candida albicans viability and virulence factors. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.03.004